Difference between revisions of "Team:TU Darmstadt/project/notebook"
| Line 100: | Line 100: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> First team meeting. Getting to know each other. First discussion about probable projects.</li> | + | <li style="font-size: 15px;"> First team meeting. Getting to know each other. First discussion about probable projects.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 118: | Line 118: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Further discussion about possible projects and time management. More research is necessary.</li> | + | <li style="font-size: 15px;"> Further discussion about possible projects and time management. More research is necessary.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 136: | Line 136: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Discussions lead to the decision for a microscope. For type and functionality research is planned.</li> | + | <li style="font-size: 15px;"> Discussions lead to the decision for a microscope. For type and functionality research is planned.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 154: | Line 154: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Ordering via IDT</li> | + | <li style="font-size: 15px;"> Ordering via IDT</li> |
<ul> | <ul> | ||
| − | <li> NodC, NodB, CDApuc and ChiA</li> | + | <li style="font-size: 15px;"> NodC, NodB, CDApuc and ChiA</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 169: | Line 169: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Discussion on type of microscope. High resolution wanted with minimum cost. More research is necessary.</li> | + | <li style="font-size: 15px;"> Discussion on type of microscope. High resolution wanted with minimum cost. More research is necessary.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 187: | Line 187: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> GoldenBraid assembly</li> | + | <li style="font-size: 15px;"> GoldenBraid assembly</li> |
<ul> | <ul> | ||
| − | <li> Cloning NodC, NodB, CDApuc and ChiA into the pUPD vector</li> | + | <li style="font-size: 15px;"> Cloning NodC, NodB, CDApuc and ChiA into the pUPD vector</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 202: | Line 202: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Decision for a holographic microscope. More research about the exact details and what is needed.</li> | + | <li style="font-size: 15px;"> Decision for a holographic microscope. More research about the exact details and what is needed.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 220: | Line 220: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Ongoing research</li> | + | <li style="font-size: 15px;"> Ongoing research</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 238: | Line 238: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> First parts like the DVD Pickup are ordered. Ongoing research.</li> | + | <li style="font-size: 15px;"> First parts like the DVD Pickup are ordered. Ongoing research.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 256: | Line 256: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Pinhole was sponsored by "Applied semiconductor optics". Ongoing research about detection system and DVD pickup control.</li> | + | <li style="font-size: 15px;"> Pinhole was sponsored by "Applied semiconductor optics". Ongoing research about detection system and DVD pickup control.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 274: | Line 274: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Cloning chiA into pSB1C3</li> | + | <li style="font-size: 15px;"> Cloning chiA into pSB1C3</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1C3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1C3</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 291: | Line 291: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Cloning nodC into pSB1K3</li> | + | <li style="font-size: 15px;"> Cloning nodC into pSB1K3</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-nodC and pSB1K3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-nodC and pSB1K3 with Xba1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1K3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1K3</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1K3-Anderson-nodC into Top10</li> | + | <li style="font-size: 15px;"> Transforming pSB1K3-Anderson-nodC into Top10</li> |
| − | <li> Over-night cultures of pSB1K3-Anderson-nodC</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1K3-Anderson-nodC</li> |
| − | <li> Plasmid extraction via Mini-prep-kit</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 314: | Line 314: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Cloning nodB into pSB1K3</li> | + | <li style="font-size: 15px;"> Cloning nodB into pSB1K3</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-nodB and pSB1K3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-nodB and pSB1K3 with Xba1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1K3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1K3</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1K3-Anderson-nodB and pUPD-Anderson-CDApuc into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1K3-Anderson-nodB and pUPD-Anderson-CDApuc into Top10 via heat-shock</li> |
| − | <li> ColonyPCR of pSB1K3-Anderson-nodB and pUPD-Anderson-CDApuc</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1K3-Anderson-nodB and pUPD-Anderson-CDApuc</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 336: | Line 336: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Research about possible software starts as well as about micromanipulators. Furthermore continuing research of the last week.</li> | + | <li style="font-size: 15px;"> Research about possible software starts as well as about micromanipulators. Furthermore continuing research of the last week.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 354: | Line 354: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Cloning chiA into pSB1C3-AraC</li> | + | <li style="font-size: 15px;"> Cloning chiA into pSB1C3-AraC</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-chiA with Nhe1 and Pst1, pSB1C3-AraC with Spe1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-chiA with Nhe1 and Pst1, pSB1C3-AraC with Spe1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1C3-AraC</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1C3-AraC</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA into Top10 via heat-shock</li> |
| − | <li> ColonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Over-night cultures of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li> |
| − | <li> Plasmid extraction via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 379: | Line 379: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Cloning nodC into pSB1C3-Anderson and pSB1C3-AraC</li> | + | <li style="font-size: 15px;"> Cloning nodC into pSB1C3-Anderson and pSB1C3-AraC</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-nodC and pSB1C3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-nodC and pSB1C3 with Xba1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1K3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1K3</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1K3-Anderson-nodC into Top10</li> | + | <li style="font-size: 15px;"> Transforming pSB1K3-Anderson-nodC into Top10</li> |
| − | <li> ColonyPCR</li> | + | <li style="font-size: 15px;"> ColonyPCR</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> |
| − | <li> Plasmid extraction via Mini-prep-kit</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 404: | Line 404: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Evaluation</li> | + | <li style="font-size: 15px;"> Evaluation</li> |
<ul> | <ul> | ||
| − | <li> Gelelectrophoresis of colonyPCR product</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis of colonyPCR product</li> |
| − | <li> Repeating the colonyPCR of pSB1C3-Anderson-nodB and pUPD-Anderson-CDApuc</li> | + | <li style="font-size: 15px;"> Repeating the colonyPCR of pSB1C3-Anderson-nodB and pUPD-Anderson-CDApuc</li> |
</ul> | </ul> | ||
| − | <li> Retransformation</li> | + | <li style="font-size: 15px;"> Retransformation</li> |
<ul> | <ul> | ||
| − | <li> Retransformation of pSB1C3-Anderson-nodB and pUPD-Anderson-CDApuc into Top10</li> | + | <li style="font-size: 15px;"> Retransformation of pSB1C3-Anderson-nodB and pUPD-Anderson-CDApuc into Top10</li> |
</ul> | </ul> | ||
| − | <li> Sequencing</li> | + | <li style="font-size: 15px;"> Sequencing</li> |
<ul> | <ul> | ||
| − | <li> Over-night cultures of pSB1C3-Anderson-nodB</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodB</li> |
| − | <li> Plasmid extraction via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit and sequencing</li> |
<ul> | <ul> | ||
| − | <li> No insert was found</li> | + | <li style="font-size: 15px;"> No insert was found</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 431: | Line 431: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Continuing research of last weeks. First ideas of DVD Pickup control and micromanipulators are acquired.</li> | + | <li style="font-size: 15px;"> Continuing research of last weeks. First ideas of DVD Pickup control and micromanipulators are acquired.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 449: | Line 449: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Cloning pSB1C3-Anderson-chiA</li> | + | <li style="font-size: 15px;"> Cloning pSB1C3-Anderson-chiA</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1C3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1C3</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation</li> | + | <li style="font-size: 15px;"> Transformation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 471: | Line 471: | ||
<article> | <article> | ||
<ul> | <ul> | ||
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> |
| − | <li> Plasmid extraction via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
| − | <li> Glycerol stocks of pSB1C3-Anderson-nodC</li> | + | <li style="font-size: 15px;"> Glycerol stocks of pSB1C3-Anderson-nodC</li> |
<ul> | <ul> | ||
| − | <li> Glycerol stocks were made of positive pSB1C3-Anderson-nodC colonies</li> | + | <li style="font-size: 15px;"> Glycerol stocks were made of positive pSB1C3-Anderson-nodC colonies</li> |
</ul> | </ul> | ||
| − | <li> Glycerol stocks of pSB1C3-AraC-nodC</li> | + | <li style="font-size: 15px;"> Glycerol stocks of pSB1C3-AraC-nodC</li> |
<ul> | <ul> | ||
| − | <li> Glycerol stocks were made of positive pSB1C3-AraC-nodC colonies</li> | + | <li style="font-size: 15px;"> Glycerol stocks were made of positive pSB1C3-AraC-nodC colonies</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 493: | Line 493: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Cloning nodB into pSB1K3</li> | + | <li style="font-size: 15px;"> Cloning nodB into pSB1K3</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-nodB and pSB1C3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-nodB and pSB1C3 with Xba1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1C3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1C3</li> |
| − | <li> PCR clean-up to</li> | + | <li style="font-size: 15px;"> PCR clean-up to</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-nodB into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-nodB into Top10 via heat-shock</li> |
<ul> | <ul> | ||
| − | <li> No colony was successful</li> | + | <li style="font-size: 15px;"> No colony was successful</li> |
</ul> | </ul> | ||
| − | <li> Cloning nodB into pSB1K3</li> | + | <li style="font-size: 15px;"> Cloning nodB into pSB1K3</li> |
| − | <li> Digesting pUPD-Anderson-nodB and pSB1C3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-nodB and pSB1C3 with Xba1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1C3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1C3</li> |
| − | <li> PCR clean-up to</li> | + | <li style="font-size: 15px;"> PCR clean-up to</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-nodB into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-nodB into Top10 via heat-shock</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 527: | Line 527: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Start working with the 3D printer. Ongoing research.</li> | + | <li style="font-size: 15px;"> Start working with the 3D printer. Ongoing research.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 545: | Line 545: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Evaluation</li> | + | <li style="font-size: 15px;"> Evaluation</li> |
<ul> | <ul> | ||
| − | <li> ColonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Over-night cultures of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li> |
| − | <li> Plasmid extraction via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 563: | Line 563: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Evaluation</li> | + | <li style="font-size: 15px;"> Evaluation</li> |
<ul> | <ul> | ||
| − | <li> Several colonyPCRs were done</li> | + | <li style="font-size: 15px;"> Several colonyPCRs were done</li> |
| − | <li> Several gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Several gelelectrophoresis</li> |
<ul> | <ul> | ||
| − | <li> They showed that the colonyPCRs did not work properly</li> | + | <li style="font-size: 15px;"> They showed that the colonyPCRs did not work properly</li> |
</ul> | </ul> | ||
| − | <li> GoldenBrain assembly</li> | + | <li style="font-size: 15px;"> GoldenBrain assembly</li> |
| − | <li> Cloning Anderson-CDApuc in pUPD</li> | + | <li style="font-size: 15px;"> Cloning Anderson-CDApuc in pUPD</li> |
</ul> | </ul> | ||
| − | <li> Transformation</li> | + | <li style="font-size: 15px;"> Transformation</li> |
<ul> | <ul> | ||
| − | <li> Transforming the pUPD-Anderson-CDApuc into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming the pUPD-Anderson-CDApuc into Top10 via heat-shock</li> |
</ul> | </ul> | ||
| − | <li> Over-night cultures of pSB1C3-Anderson-nodB and pSB1K3-Anderson-nodB</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodB and pSB1K3-Anderson-nodB</li> |
| − | <li> DNA-preparation</li> | + | <li style="font-size: 15px;"> DNA-preparation</li> |
<ul> | <ul> | ||
| − | <li> Plasmid extraction of pSB1C3-Anderson-nodB and pSB1K3-Anderson-nodB via Mini-prep Kit</li> | + | <li style="font-size: 15px;"> Plasmid extraction of pSB1C3-Anderson-nodB and pSB1K3-Anderson-nodB via Mini-prep Kit</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 593: | Line 593: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> 3D printer crashed and needs repair. Ongoing research.</li> | + | <li style="font-size: 15px;"> 3D printer crashed and needs repair. Ongoing research.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 611: | Line 611: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Retransformation and evaluation</li> | + | <li style="font-size: 15px;"> Retransformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Retransforming pSB1C3-AraC-chiA into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Retransforming pSB1C3-AraC-chiA into Top10 via heat-shock</li> |
| − | <li> ColonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Over-night cultures</li> | + | <li style="font-size: 15px;"> Over-night cultures</li> |
| − | <li> Plasmid extraction via Mini-prep-kit</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit</li> |
</ul> | </ul> | ||
| − | <li> Detection of a point mutation in pSB1C3-Anderson-ChiA</li> | + | <li style="font-size: 15px;"> Detection of a point mutation in pSB1C3-Anderson-ChiA</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 632: | Line 632: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> DNA-preparation</li> | + | <li style="font-size: 15px;"> DNA-preparation</li> |
<ul> | <ul> | ||
| − | <li> Plasmid extraction of pUPD-Anderson-CDApuc via Mini-prep Kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction of pUPD-Anderson-CDApuc via Mini-prep Kit and sequencing</li> |
</ul> | </ul> | ||
| − | <li> Cloning</li> | + | <li style="font-size: 15px;"> Cloning</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-nodB with Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-nodB with Pst1</li> |
| − | <li> PCR clean-up</li> | + | <li style="font-size: 15px;"> PCR clean-up</li> |
| − | <li> Digesting pUPD-Anderson-nodB with Pst1 again</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-nodB with Pst1 again</li> |
| − | <li> Ligation of nodB and pSB1C3 with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation of nodB and pSB1C3 with T4 Ligase</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 654: | Line 654: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Still fixing 3D printer. Ongoing research.</li> | + | <li style="font-size: 15px;"> Still fixing 3D printer. Ongoing research.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 672: | Line 672: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-AraC-chiA into BL21 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-AraC-chiA into BL21 via heat-shock</li> |
| − | <li> ColonyPCR of pSB1C3-AraC-chiA</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1C3-AraC-chiA</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 689: | Line 689: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Cloning nodC into pSB1K3</li> | + | <li style="font-size: 15px;"> Cloning nodC into pSB1K3</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-nodC with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-nodC with Xba1 and Pst1</li> |
| − | <li> Digesting pSB1K3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pSB1K3 with Xba1 and Pst1</li> |
| − | <li> Gelelectrophoresis and gelextraction</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis and gelextraction</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Cloning nodC into pSB1C3</li> | + | <li style="font-size: 15px;"> Cloning nodC into pSB1C3</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-nodC with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-nodC with Xba1 and Pst1</li> |
| − | <li> Digesting pSB1C3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pSB1C3 with Xba1 and Pst1</li> |
| − | <li> Gelelectrophoresis and gelextraction</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis and gelextraction</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Cloning nodC into pSB1C3-AraC</li> | + | <li style="font-size: 15px;"> Cloning nodC into pSB1C3-AraC</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-nodC with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-nodC with Xba1 and Pst1</li> |
| − | <li> Digesting pSb1C3-AraC with Spe1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pSb1C3-AraC with Spe1 and Pst1</li> |
| − | <li> Gelelectrophoresis and gelextraction</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis and gelextraction</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation</li> | + | <li style="font-size: 15px;"> Transformation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1K3-Anderson-nodC, pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1K3-Anderson-nodC, pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into Top10 via heat-shock</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 725: | Line 725: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Transformation</li> | + | <li style="font-size: 15px;"> Transformation</li> |
<ul> | <ul> | ||
| − | <li> Transforming the pSB1C3-nodB into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming the pSB1C3-nodB into Top10 via heat-shock</li> |
</ul> | </ul> | ||
| − | <li> Evaluation</li> | + | <li style="font-size: 15px;"> Evaluation</li> |
<ul> | <ul> | ||
| − | <li> ColonyPCR of pSB1C3-nodB</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1C3-nodB</li> |
| − | <li> Over-night cultures of pSB1C3-nodB</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-nodB</li> |
| − | <li> Plasmid extraction of pSB1C3-nodB via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction of pSB1C3-nodB via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
| − | <li> Cloning CDApuc into pSB1C3 and pSB1K3 with and without Anderson-promotor</li> | + | <li style="font-size: 15px;"> Cloning CDApuc into pSB1C3 and pSB1K3 with and without Anderson-promotor</li> |
| − | <li> Transformation</li> | + | <li style="font-size: 15px;"> Transformation</li> |
<ul> | <ul> | ||
| − | <li> Transforming the pSB1C3-CDApuc, pSB1K3-CDApuc, pSB1C3-Anderson-CDApuc and pSB1K3-Anderson-CDApuc into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming the pSB1C3-CDApuc, pSB1K3-CDApuc, pSB1C3-Anderson-CDApuc and pSB1K3-Anderson-CDApuc into Top10 via heat-shock</li> |
</ul> | </ul> | ||
| − | <li> Over-night cultures of pSB1C3-CDApuc, pSB1K3-CDApuc, pSB1C3-Anderson-CDApuc and pSB1K3-Anderson-CDApuc</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-CDApuc, pSB1K3-CDApuc, pSB1C3-Anderson-CDApuc and pSB1K3-Anderson-CDApuc</li> |
| − | <li> DNA-preparation</li> | + | <li style="font-size: 15px;"> DNA-preparation</li> |
<ul> | <ul> | ||
| − | <li> Plasmid extraction of pSB1C3-CDApuc and pSB1K3-CDApuc via Mini-prep Kit</li> | + | <li style="font-size: 15px;"> Plasmid extraction of pSB1C3-CDApuc and pSB1K3-CDApuc via Mini-prep Kit</li> |
</ul> | </ul> | ||
| − | <li> Gelelectrophoresis of pSB1C3-CDApuc and pSB1K3-CDApuc</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis of pSB1C3-CDApuc and pSB1K3-CDApuc</li> |
| − | <li> Transformation (repeat)</li> | + | <li style="font-size: 15px;"> Transformation (repeat)</li> |
<ul> | <ul> | ||
| − | <li> Transforming the pSB1C3-nodB into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming the pSB1C3-nodB into Top10 via heat-shock</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 761: | Line 761: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Learning about CAD and working on manipulators. Continuing last week?s work.</li> | + | <li style="font-size: 15px;"> Learning about CAD and working on manipulators. Continuing last week?s work.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 779: | Line 779: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Over-night cultures of pSB1C3-AraC-chiA and empty BL21 cells</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-AraC-chiA and empty BL21 cells</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
<ul> | <ul> | ||
| − | <li> Induction with arabinose at OD=0.6</li> | + | <li style="font-size: 15px;"> Induction with arabinose at OD=0.6</li> |
<ul> | <ul> | ||
| − | <li> Taking sample1 before induction</li> | + | <li style="font-size: 15px;"> Taking sample1 before induction</li> |
| − | <li> Taking sample2 three hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample2 three hours after induction</li> |
| − | <li> Taking sample3 24 hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample3 24 hours after induction</li> |
</ul> | </ul> | ||
| − | <li> Store samples at -20 degree Celsius</li> | + | <li style="font-size: 15px;"> Store samples at -20 degree Celsius</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 801: | Line 801: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Evaluation of transformations</li> | + | <li style="font-size: 15px;"> Evaluation of transformations</li> |
<ul> | <ul> | ||
| − | <li> colonyPCR of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> | + | <li style="font-size: 15px;"> colonyPCR of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21</li> |
| − | <li> Plasmid extraction via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 819: | Line 819: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Cloning nodB in pSB1K3</li> | + | <li style="font-size: 15px;"> Cloning nodB in pSB1K3</li> |
<ul> | <ul> | ||
| − | <li> Digesting pSB1C3-nodB and pSB1K3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pSB1C3-nodB and pSB1K3 with Xba1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1K3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1K3</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation</li> | + | <li style="font-size: 15px;"> Transformation</li> |
<ul> | <ul> | ||
| − | <li> Transforming the pSB1K3-nodB into Top10</li> | + | <li style="font-size: 15px;"> Transforming the pSB1K3-nodB into Top10</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 840: | Line 840: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Continuing last week's work</li> | + | <li style="font-size: 15px;"> Continuing last week's work</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 858: | Line 858: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Over-night cultures of pSB1C3-AraC-chiA and empty BL21 cells</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-AraC-chiA and empty BL21 cells</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
<ul> | <ul> | ||
| − | <li> Induction with arabinose at OD=0.6</li> | + | <li style="font-size: 15px;"> Induction with arabinose at OD=0.6</li> |
<ul> | <ul> | ||
| − | <li> Taking sample1 before induction</li> | + | <li style="font-size: 15px;"> Taking sample1 before induction</li> |
| − | <li> Taking sample2 three hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample2 three hours after induction</li> |
| − | <li> Taking sample3 24 hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample3 24 hours after induction</li> |
</ul> | </ul> | ||
| − | <li> Store samples at -20 degree Celsius</li> | + | <li style="font-size: 15px;"> Store samples at -20 degree Celsius</li> |
</ul> | </ul> | ||
| − | <li> SDS-Page</li> | + | <li style="font-size: 15px;"> SDS-Page</li> |
<ul> | <ul> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-AraC-chiA into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-AraC-chiA into Top10 via heat-shock</li> |
| − | <li> ColonyPCR of pSB1C3-AraC-chiA</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1C3-AraC-chiA</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 890: | Line 890: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 904: | Line 904: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Evaluation</li> | + | <li style="font-size: 15px;"> Evaluation</li> |
<ul> | <ul> | ||
| − | <li> ColonyPCR of pSB1K3-Anderson-nodB</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1K3-Anderson-nodB</li> |
| − | <li> Plasmid extraction of pSB1C3-Anderson-CDApuc, pSB1K3-Anderson-CDApuc and pSB1C3-CDApuc via Mini-prep Kit</li> | + | <li style="font-size: 15px;"> Plasmid extraction of pSB1C3-Anderson-CDApuc, pSB1K3-Anderson-CDApuc and pSB1C3-CDApuc via Mini-prep Kit</li> |
| − | <li> Restriction digestion and gelelectrophoresis of pSB1C3-Anderson-CDApuc, pSB1K3-Anderson-CDApuc and pSB1C3-CDApuc</li> | + | <li style="font-size: 15px;"> Restriction digestion and gelelectrophoresis of pSB1C3-Anderson-CDApuc, pSB1K3-Anderson-CDApuc and pSB1C3-CDApuc</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 921: | Line 921: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Continuing last week's work</li> | + | <li style="font-size: 15px;"> Continuing last week's work</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 939: | Line 939: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-AraC-chiA into BL21 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-AraC-chiA into BL21 via heat-shock</li> |
| − | <li> ColonyPCR of pSB1C3-AraC-chiA</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1C3-AraC-chiA</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Over-night cultures of pSB1C3-AraC-chiA and empty BL21 cells</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-AraC-chiA and empty BL21 cells</li> |
</ul> | </ul> | ||
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
<ul> | <ul> | ||
| − | <li> Induction with arabinose at OD=0.6</li> | + | <li style="font-size: 15px;"> Induction with arabinose at OD=0.6</li> |
<ul> | <ul> | ||
| − | <li> Taking sample1 before induction</li> | + | <li style="font-size: 15px;"> Taking sample1 before induction</li> |
| − | <li> Taking sample2 three hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample2 three hours after induction</li> |
| − | <li> Taking sample3 24 hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample3 24 hours after induction</li> |
</ul> | </ul> | ||
| − | <li> Store samples at -20 degree Celsius</li> | + | <li style="font-size: 15px;"> Store samples at -20 degree Celsius</li> |
</ul> | </ul> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
<ul> | <ul> | ||
| − | <li> It showed the expected band at 46,5 kDa</li> | + | <li style="font-size: 15px;"> It showed the expected band at 46,5 kDa</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 971: | Line 971: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into BL21 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into BL21 via heat-shock</li> |
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21 cells</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21 cells</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
<ul> | <ul> | ||
| − | <li> Induction with arabinose at OD=0.6</li> | + | <li style="font-size: 15px;"> Induction with arabinose at OD=0.6</li> |
<ul> | <ul> | ||
| − | <li> Taking sample1 before induction</li> | + | <li style="font-size: 15px;"> Taking sample1 before induction</li> |
| − | <li> Taking sample2 three hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample2 three hours after induction</li> |
| − | <li> Taking sample3 six hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample3 six hours after induction</li> |
| − | <li> Taking sample4 the next day</li> | + | <li style="font-size: 15px;"> Taking sample4 the next day</li> |
</ul> | </ul> | ||
| − | <li> Store samples at -20 degree Celsius</li> | + | <li style="font-size: 15px;"> Store samples at -20 degree Celsius</li> |
</ul> | </ul> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 997: | Line 997: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> 3D printer is fixed. Deciding on a raspberry pie cam for detecting the hologram. First control for DVD pick up is build, but breaks down after several tests. DIYouware PCB board is investigated. Collaboration with iGEM Team York. We send them one of our pickups.</li> | + | <li style="font-size: 15px;"> 3D printer is fixed. Deciding on a raspberry pie cam for detecting the hologram. First control for DVD pick up is build, but breaks down after several tests. DIYouware PCB board is investigated. Collaboration with iGEM Team York. We send them one of our pickups.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,010: | Line 1,010: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> After consultation with an expert the CDApuc was deemed incompatible with the project</li> | + | <li style="font-size: 15px;"> After consultation with an expert the CDApuc was deemed incompatible with the project</li> |
| − | <li> Evaluation</li> | + | <li style="font-size: 15px;"> Evaluation</li> |
<ul> | <ul> | ||
| − | <li> Plasmid extraction via Mini-prep kit and sequencing,</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep kit and sequencing,</li> |
</ul> | </ul> | ||
| − | <li> Glycerol stocks</li> | + | <li style="font-size: 15px;"> Glycerol stocks</li> |
<ul> | <ul> | ||
| − | <li> Glycerol stocks of pSB1K3-Anderson-nodB were made</li> | + | <li style="font-size: 15px;"> Glycerol stocks of pSB1K3-Anderson-nodB were made</li> |
</ul> | </ul> | ||
| − | <li> Over-night cultures of pSB1C3-Anderson-nodB, pSB1C3-Anderson-nodB and empty Top10</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodB, pSB1C3-Anderson-nodB and empty Top10</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
<ul> | <ul> | ||
| − | <li> No induction needed as the Anderson-promotor is constitutive</li> | + | <li style="font-size: 15px;"> No induction needed as the Anderson-promotor is constitutive</li> |
| − | <li> Taking sample1 at OD=0.6</li> | + | <li style="font-size: 15px;"> Taking sample1 at OD=0.6</li> |
| − | <li> Taking sample2 three hours later</li> | + | <li style="font-size: 15px;"> Taking sample2 three hours later</li> |
| − | <li> Taking sample3 six hours later</li> | + | <li style="font-size: 15px;"> Taking sample3 six hours later</li> |
| − | <li> Taking sample4 24 hours later</li> | + | <li style="font-size: 15px;"> Taking sample4 24 hours later</li> |
| − | <li> Store samples at -20 degree Celsius</li> | + | <li style="font-size: 15px;"> Store samples at -20 degree Celsius</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,045: | Line 1,045: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Cloning chiA into pSB1C3</li> | + | <li style="font-size: 15px;"> Cloning chiA into pSB1C3</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1C3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1C3</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation</li> | + | <li style="font-size: 15px;"> Transformation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,066: | Line 1,066: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,080: | Line 1,080: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,093: | Line 1,093: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Pi Cam is ordered. Learning PCB boards. Printing parts for adjustment of pinhole and DVD pickup. Ongoing research about software.</li> | + | <li style="font-size: 15px;"> Pi Cam is ordered. Learning PCB boards. Printing parts for adjustment of pinhole and DVD pickup. Ongoing research about software.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,111: | Line 1,111: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Evaluation of transformation</li> | + | <li style="font-size: 15px;"> Evaluation of transformation</li> |
<ul> | <ul> | ||
| − | <li> ColonyPCR of pSB1C3-Anderson-chiA</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1C3-Anderson-chiA</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Over-night cultures of pSB1C3-Anderson-chiA</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-chiA</li> |
| − | <li> Plasmid extraction via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
| − | <li> The mutation still remains in the construct</li> | + | <li style="font-size: 15px;"> The mutation still remains in the construct</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,131: | Line 1,131: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Transformation</li> | + | <li style="font-size: 15px;"> Transformation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into BL21 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into BL21 via heat-shock</li> |
</ul> | </ul> | ||
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21 cells</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21 cells</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
<ul> | <ul> | ||
| − | <li> Induction with arabinose at OD=0.6</li> | + | <li style="font-size: 15px;"> Induction with arabinose at OD=0.6</li> |
<ul> | <ul> | ||
| − | <li> Taking sample1 before induction</li> | + | <li style="font-size: 15px;"> Taking sample1 before induction</li> |
| − | <li> Taking sample2 three hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample2 three hours after induction</li> |
| − | <li> Taking sample3 six hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample3 six hours after induction</li> |
| − | <li> Taking sample4 the next day</li> | + | <li style="font-size: 15px;"> Taking sample4 the next day</li> |
</ul> | </ul> | ||
| − | <li> Store samples at -20 degree Celsius</li> | + | <li style="font-size: 15px;"> Store samples at -20 degree Celsius</li> |
</ul> | </ul> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,160: | Line 1,160: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
| − | <li> Transformation</li> | + | <li style="font-size: 15px;"> Transformation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pUPD-nodB into BL21 cells</li> | + | <li style="font-size: 15px;"> Transforming pUPD-nodB into BL21 cells</li> |
<ul> | <ul> | ||
| − | <li> The pUPD vector does contain the T7-promotor sequence, which was not used before</li> | + | <li style="font-size: 15px;"> The pUPD vector does contain the T7-promotor sequence, which was not used before</li> |
<ul> | <ul> | ||
| − | <li> pUPD-T7-nodB is used for detection because the T7-promotor is inducible with IPTG</li> | + | <li style="font-size: 15px;"> pUPD-T7-nodB is used for detection because the T7-promotor is inducible with IPTG</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,180: | Line 1,180: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> First try of acquiring holograms. Probably intensity to low, further improvement necessary.</li> | + | <li style="font-size: 15px;"> First try of acquiring holograms. Probably intensity to low, further improvement necessary.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,198: | Line 1,198: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Cloning Anderson-chiA into pSB1C3</li> | + | <li style="font-size: 15px;"> Cloning Anderson-chiA into pSB1C3</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1C3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1C3</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li> |
| − | <li> ColonyPCR of pSB1C3-Anderson-chiA</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1C3-Anderson-chiA</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Over-night cultures of pSB1C3-Anderson-chiA</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-chiA</li> |
| − | <li> Plasmid extraction via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
| − | <li> The mutation still remains in the construct</li> | + | <li style="font-size: 15px;"> The mutation still remains in the construct</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,225: | Line 1,225: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21 cells</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21 cells</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
<ul> | <ul> | ||
| − | <li> Induction with arabinose at OD=0.6</li> | + | <li style="font-size: 15px;"> Induction with arabinose at OD=0.6</li> |
<ul> | <ul> | ||
| − | <li> Taking sample1 before induction</li> | + | <li style="font-size: 15px;"> Taking sample1 before induction</li> |
| − | <li> Taking sample2 three hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample2 three hours after induction</li> |
| − | <li> Taking sample3 six hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample3 six hours after induction</li> |
| − | <li> Taking sample4 the next day</li> | + | <li style="font-size: 15px;"> Taking sample4 the next day</li> |
</ul> | </ul> | ||
| − | <li> Store samples at -20 degree Celsius</li> | + | <li style="font-size: 15px;"> Store samples at -20 degree Celsius</li> |
</ul> | </ul> | ||
| − | <li> A SDS-Page was performed but showed no satisfactory results</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed but showed no satisfactory results</li> |
| − | <li> Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into BL21 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into BL21 via heat-shock</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,251: | Line 1,251: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Over-night cultures of pUPD-T7-nodB and empty BL21 cells</li> | + | <li style="font-size: 15px;"> Over-night cultures of pUPD-T7-nodB and empty BL21 cells</li> |
| − | <li> Growing a main cultur</li> | + | <li style="font-size: 15px;"> Growing a main cultur</li> |
<ul> | <ul> | ||
| − | <li> Induction with IPTG at OD=0.6</li> | + | <li style="font-size: 15px;"> Induction with IPTG at OD=0.6</li> |
<ul> | <ul> | ||
| − | <li> Taking sample1 before induction</li> | + | <li style="font-size: 15px;"> Taking sample1 before induction</li> |
| − | <li> Taking sample2 three hours later</li> | + | <li style="font-size: 15px;"> Taking sample2 three hours later</li> |
| − | <li> Taking sample3 six hours later</li> | + | <li style="font-size: 15px;"> Taking sample3 six hours later</li> |
| − | <li> Taking sample4 24 hours later</li> | + | <li style="font-size: 15px;"> Taking sample4 24 hours later</li> |
</ul> | </ul> | ||
| − | <li> Store samples at -20 degree Celsius</li> | + | <li style="font-size: 15px;"> Store samples at -20 degree Celsius</li> |
</ul> | </ul> | ||
| − | <li> A SDS-Page was performed and repeated</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed and repeated</li> |
<ul> | <ul> | ||
| − | <li> It showed the expected band at 23 kDa</li> | + | <li style="font-size: 15px;"> It showed the expected band at 23 kDa</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,278: | Line 1,278: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Implementing an self-made millimetre sized pinhole to increase intensity. Pictures were taken. Holopy is chosen as suitable software.</li> | + | <li style="font-size: 15px;"> Implementing an self-made millimetre sized pinhole to increase intensity. Pictures were taken. Holopy is chosen as suitable software.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,296: | Line 1,296: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Cloning Anderson-chiA into pSB1C3</li> | + | <li style="font-size: 15px;"> Cloning Anderson-chiA into pSB1C3</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1C3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1C3</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li> |
| − | <li> ColonyPCR of pSB1C3-Anderson-chiA</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1C3-Anderson-chiA</li> |
| − | <li> Gelelektrophoresis</li> | + | <li style="font-size: 15px;"> Gelelektrophoresis</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,319: | Line 1,319: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and BL21 cells</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and BL21 cells</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
<ul> | <ul> | ||
| − | <li> Induction with arabinose at OD=0.6</li> | + | <li style="font-size: 15px;"> Induction with arabinose at OD=0.6</li> |
<ul> | <ul> | ||
| − | <li> Taking sample1 before induction</li> | + | <li style="font-size: 15px;"> Taking sample1 before induction</li> |
| − | <li> Taking sample2 three hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample2 three hours after induction</li> |
| − | <li> Take sample3 six hours after induction</li> | + | <li style="font-size: 15px;"> Take sample3 six hours after induction</li> |
| − | <li> Take sample4 the next day</li> | + | <li style="font-size: 15px;"> Take sample4 the next day</li> |
</ul> | </ul> | ||
| − | <li> Store samples at -20 degree Celsius</li> | + | <li style="font-size: 15px;"> Store samples at -20 degree Celsius</li> |
</ul> | </ul> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
| − | <li> Determinating the best medium</li> | + | <li style="font-size: 15px;"> Determinating the best medium</li> |
<ul> | <ul> | ||
| − | <li> Media tested: LB, TB and SOC</li> | + | <li style="font-size: 15px;"> Media tested: LB, TB and SOC</li> |
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,348: | Line 1,348: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> ordering PCB boards and all parts necessary for it. Further work on micromanipulators, learning about flexing bearings. Research about the full functionality of the holopy software is carried out. York do not want to use DVD pickup due to safety reasons.</li> | + | <li style="font-size: 15px;"> ordering PCB boards and all parts necessary for it. Further work on micromanipulators, learning about flexing bearings. Research about the full functionality of the holopy software is carried out. York do not want to use DVD pickup due to safety reasons.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,366: | Line 1,366: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Cloning pSB1C3-Anderson-chiA</li> | + | <li style="font-size: 15px;"> Cloning pSB1C3-Anderson-chiA</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li> |
| − | <li> Gelelektrophoresis and gelextraction</li> | + | <li style="font-size: 15px;"> Gelelektrophoresis and gelextraction</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-chiA into Top10</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-chiA into Top10</li> |
| − | <li> ColonyPCR</li> | + | <li style="font-size: 15px;"> ColonyPCR</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Over-night cultures of pSB1C3-Anderson-chiA</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-chiA</li> |
| − | <li> Plasmid extraction via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
| − | <li> The mutation still remains in the construct</li> | + | <li style="font-size: 15px;"> The mutation still remains in the construct</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,393: | Line 1,393: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Determinating the best medium</li> | + | <li style="font-size: 15px;"> Determinating the best medium</li> |
<ul> | <ul> | ||
| − | <li> Media tested: LB, TB and SOC</li> | + | <li style="font-size: 15px;"> Media tested: LB, TB and SOC</li> |
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> |
| − | <li> tracing cell-growth over 48 hours</li> | + | <li style="font-size: 15px;"> tracing cell-growth over 48 hours</li> |
</ul> | </ul> | ||
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21 cells</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21 cells</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
<ul> | <ul> | ||
| − | <li> Induction with arabinose at OD=0.6</li> | + | <li style="font-size: 15px;"> Induction with arabinose at OD=0.6</li> |
<ul> | <ul> | ||
| − | <li> Taking sample1 before induction</li> | + | <li style="font-size: 15px;"> Taking sample1 before induction</li> |
| − | <li> Taking sample2 three hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample2 three hours after induction</li> |
| − | <li> Taking sample3 six hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample3 six hours after induction</li> |
| − | <li> Taking sample4 the next day</li> | + | <li style="font-size: 15px;"> Taking sample4 the next day</li> |
</ul> | </ul> | ||
| − | <li> Store samples at -20 degree Celsius</li> | + | <li style="font-size: 15px;"> Store samples at -20 degree Celsius</li> |
</ul> | </ul> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,424: | Line 1,424: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> First flexible bearing manipulators are printed and tested. Discussion about what to see on the first images. Ongoing work with the software.</li> | + | <li style="font-size: 15px;"> First flexible bearing manipulators are printed and tested. Discussion about what to see on the first images. Ongoing work with the software.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,442: | Line 1,442: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
| − | <li> Mutagenesis PCR for the purpose of purification</li> | + | <li style="font-size: 15px;"> Mutagenesis PCR for the purpose of purification</li> |
<ul> | <ul> | ||
| − | <li> Site-directed-mutagenesis: His-Tag to pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> | + | <li style="font-size: 15px;"> Site-directed-mutagenesis: His-Tag to pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,458: | Line 1,458: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> GoldenBraid assembly</li> | + | <li style="font-size: 15px;"> GoldenBraid assembly</li> |
<ul> | <ul> | ||
| − | <li> Cloning CODvc into pUPD</li> | + | <li style="font-size: 15px;"> Cloning CODvc into pUPD</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pUPD-T7-CODvc into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pUPD-T7-CODvc into Top10 via heat-shock</li> |
| − | <li> Over-night cultures of pUPD-T7-CODvc</li> | + | <li style="font-size: 15px;"> Over-night cultures of pUPD-T7-CODvc</li> |
| − | <li> Plasmid extraction via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,479: | Line 1,479: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Chitosan solved with acetic acid</li> | + | <li style="font-size: 15px;"> Chitosan solved with acetic acid</li> |
<ul> | <ul> | ||
| − | <li> Different chitosan/acetic acid concentration were tested</li> | + | <li style="font-size: 15px;"> Different chitosan/acetic acid concentration were tested</li> |
| − | <li> in 100 % Milli-Q H2O</li> | + | <li style="font-size: 15px;"> in 100 % Milli-Q H2O</li> |
| − | <li> 1%Chitosan - 0,5 % acetic acid</li> | + | <li style="font-size: 15px;"> 1%Chitosan - 0,5 % acetic acid</li> |
| − | <li> 1%Chitosan - 1 % acetic acid</li> | + | <li style="font-size: 15px;"> 1%Chitosan - 1 % acetic acid</li> |
| − | <li> 1%Chitosan - 1,5 % acetic acid</li> | + | <li style="font-size: 15px;"> 1%Chitosan - 1,5 % acetic acid</li> |
| − | <li> 1%Chitosan - 2 % acetic acid</li> | + | <li style="font-size: 15px;"> 1%Chitosan - 2 % acetic acid</li> |
| − | <li> 1,5%Chitosan - 0,5 % acetic acid</li> | + | <li style="font-size: 15px;"> 1,5%Chitosan - 0,5 % acetic acid</li> |
| − | <li> 1,5%Chitosan - 1 % acetic acid</li> | + | <li style="font-size: 15px;"> 1,5%Chitosan - 1 % acetic acid</li> |
| − | <li> 1,5%Chitosan - 1,5 % acetic acid</li> | + | <li style="font-size: 15px;"> 1,5%Chitosan - 1,5 % acetic acid</li> |
| − | <li> 1,5%Chitosan - 2 % acetic acid</li> | + | <li style="font-size: 15px;"> 1,5%Chitosan - 2 % acetic acid</li> |
| − | <li> 2%Chitosan - 0,5 % acetic acid</li> | + | <li style="font-size: 15px;"> 2%Chitosan - 0,5 % acetic acid</li> |
| − | <li> 2%Chitosan - 1 % acetic acid</li> | + | <li style="font-size: 15px;"> 2%Chitosan - 1 % acetic acid</li> |
| − | <li> 2%Chitosan - 1,5 % acetic acid</li> | + | <li style="font-size: 15px;"> 2%Chitosan - 1,5 % acetic acid</li> |
| − | <li> 2%Chitosan - 2 % acetic acid</li> | + | <li style="font-size: 15px;"> 2%Chitosan - 2 % acetic acid</li> |
| − | <li> 3%Chitosan - 0,5 % acetic acid</li> | + | <li style="font-size: 15px;"> 3%Chitosan - 0,5 % acetic acid</li> |
| − | <li> 3%Chitosan - 1 % acetic acid</li> | + | <li style="font-size: 15px;"> 3%Chitosan - 1 % acetic acid</li> |
<ul> | <ul> | ||
| − | <li> It is possible to solve chitosan, but it needs time and mechanical stirring, the more acid the visose the solution gets</li> | + | <li style="font-size: 15px;"> It is possible to solve chitosan, but it needs time and mechanical stirring, the more acid the visose the solution gets</li> |
</ul> | </ul> | ||
| − | <li> Chitosan-Agarose Hydrogels</li> | + | <li style="font-size: 15px;"> Chitosan-Agarose Hydrogels</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Agarose 1%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Agarose 1%</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Agarose 2%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Agarose 2%</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Agarose 3%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Agarose 3%</li> |
| − | <li> Chitosan 2%, Acetic acid 1%, Agarose 1%</li> | + | <li style="font-size: 15px;"> Chitosan 2%, Acetic acid 1%, Agarose 1%</li> |
| − | <li> Chitosan 2%, Acetic acid 1%, Agarose 2%</li> | + | <li style="font-size: 15px;"> Chitosan 2%, Acetic acid 1%, Agarose 2%</li> |
| − | <li> Chitosan 2%, Acetic acid 1%, Agarose 3%</li> | + | <li style="font-size: 15px;"> Chitosan 2%, Acetic acid 1%, Agarose 3%</li> |
</ul> | </ul> | ||
| − | <li> Chitosan was dissolved in water and 1% acetic acid, then agarose is added and heated.</li> | + | <li style="font-size: 15px;"> Chitosan was dissolved in water and 1% acetic acid, then agarose is added and heated.</li> |
<ul> | <ul> | ||
| − | <li> Chitosan is slighlty soluble.</li> | + | <li style="font-size: 15px;"> Chitosan is slighlty soluble.</li> |
</ul> | </ul> | ||
| − | <li> New approach:</li> | + | <li style="font-size: 15px;"> New approach:</li> |
<ul> | <ul> | ||
| − | <li> first Agarose was dissolved in water by heating. Then acetic acid and chitosan were added -> better results, better soluble</li> | + | <li style="font-size: 15px;"> first Agarose was dissolved in water by heating. Then acetic acid and chitosan were added -> better results, better soluble</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Agarose 1%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Agarose 1%</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Agarose 3%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Agarose 3%</li> |
<ul> | <ul> | ||
| − | <li> With 1%: promising</li> | + | <li style="font-size: 15px;"> With 1%: promising</li> |
</ul> | </ul> | ||
| − | <li> Chitosan-Agar Hydrogels</li> | + | <li style="font-size: 15px;"> Chitosan-Agar Hydrogels</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Agar 1%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Agar 1%</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Agar 2%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Agar 2%</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Agar 3%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Agar 3%</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,536: | Line 1,536: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Further work on bearings and software.</li> | + | <li style="font-size: 15px;"> Further work on bearings and software.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,554: | Line 1,554: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Mutagenesis PCR</li> | + | <li style="font-size: 15px;"> Mutagenesis PCR</li> |
<ul> | <ul> | ||
| − | <li> Phosphorylation of mutagenesis PCR batch</li> | + | <li style="font-size: 15px;"> Phosphorylation of mutagenesis PCR batch</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
| − | <li> DPN1 digestion</li> | + | <li style="font-size: 15px;"> DPN1 digestion</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
</ul> | </ul> | ||
| − | <li> Transformation into Top10 and evaluation</li> | + | <li style="font-size: 15px;"> Transformation into Top10 and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His into Top10 via heat-shock</li> |
| − | <li> ColonyPCR</li> | + | <li style="font-size: 15px;"> ColonyPCR</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His</li> |
| − | <li> Plasmid extraction via Mini-prep-kit</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His into BL21 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His into BL21 via heat-shock</li> |
| − | <li> colonyPCR of pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His</li> | + | <li style="font-size: 15px;"> colonyPCR of pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His</li> |
| − | <li> Plasmid extraction via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,588: | Line 1,588: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Mutagenese PCR</li> | + | <li style="font-size: 15px;"> Mutagenese PCR</li> |
<ul> | <ul> | ||
| − | <li> PCR with His-tag primers for mutagenesis PCR</li> | + | <li style="font-size: 15px;"> PCR with His-tag primers for mutagenesis PCR</li> |
| − | <li> Phosphorylation of mutagenesis PCR batch</li> | + | <li style="font-size: 15px;"> Phosphorylation of mutagenesis PCR batch</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
| − | <li> DPN1 digestion</li> | + | <li style="font-size: 15px;"> DPN1 digestion</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-NodB-His and pUPD-NodB-His into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-NodB-His and pUPD-NodB-His into Top10 via heat-shock</li> |
| − | <li> ColonyPCR of</li> | + | <li style="font-size: 15px;"> ColonyPCR of</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
</ul> | </ul> | ||
| − | <li> Mutagenese PCR (repeat)</li> | + | <li style="font-size: 15px;"> Mutagenese PCR (repeat)</li> |
<ul> | <ul> | ||
| − | <li> PCR with His-tag primers for mutagenesis PCR</li> | + | <li style="font-size: 15px;"> PCR with His-tag primers for mutagenesis PCR</li> |
| − | <li> Phosphorylation of mutagenesis PCR batch</li> | + | <li style="font-size: 15px;"> Phosphorylation of mutagenesis PCR batch</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
| − | <li> DPN1 digestion</li> | + | <li style="font-size: 15px;"> DPN1 digestion</li> |
</ul> | </ul> | ||
| − | <li> Transformation</li> | + | <li style="font-size: 15px;"> Transformation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-NodB-His and pUPD-NodB-His into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-NodB-His and pUPD-NodB-His into Top10 via heat-shock</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,623: | Line 1,623: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> DAB Gel</li> | + | <li style="font-size: 15px;"> DAB Gel</li> |
<ul> | <ul> | ||
| − | <li> Hydroxyethylcellulose 2,5</li> | + | <li style="font-size: 15px;"> Hydroxyethylcellulose 2,5</li> |
| − | <li> Glycerol 10%</li> | + | <li style="font-size: 15px;"> Glycerol 10%</li> |
| − | <li> Milli-Q H2O 87,5%</li> | + | <li style="font-size: 15px;"> Milli-Q H2O 87,5%</li> |
| − | <li> no swelling</li> | + | <li style="font-size: 15px;"> no swelling</li> |
<ul> | <ul> | ||
| − | <li> Hydroxyethylcellulose was not "Hydroxyethylcellulose 10000", but with a lower viscosity, so the gel did not form a stable gel</li> | + | <li style="font-size: 15px;"> Hydroxyethylcellulose was not "Hydroxyethylcellulose 10000", but with a lower viscosity, so the gel did not form a stable gel</li> |
</ul> | </ul> | ||
| − | <li> Order of mixing was determed;</li> | + | <li style="font-size: 15px;"> Order of mixing was determed;</li> |
| − | <li> 1) 2% Chitosan, than 97,5% Milli-Q water, than 0,5% acetic acid</li> | + | <li style="font-size: 15px;"> 1) 2% Chitosan, than 97,5% Milli-Q water, than 0,5% acetic acid</li> |
| − | <li> 2) 2% Chitosan, than 50% Milli-Q water, than 0,5% acetic acid, than 47,5% Milli-Q water</li> | + | <li style="font-size: 15px;"> 2) 2% Chitosan, than 50% Milli-Q water, than 0,5% acetic acid, than 47,5% Milli-Q water</li> |
| − | <li> 3) 2% Chitosan, than 0,5% acetic acid, than 98% Milli-Q water</li> | + | <li style="font-size: 15px;"> 3) 2% Chitosan, than 0,5% acetic acid, than 98% Milli-Q water</li> |
| − | <li> 4) 97,5% Milli-Q water mixed with 0,5% acetic acid, than 2% Chitosan</li> | + | <li style="font-size: 15px;"> 4) 97,5% Milli-Q water mixed with 0,5% acetic acid, than 2% Chitosan</li> |
</ul> | </ul> | ||
| − | <li> Chitosan-Agarose Hydrogels</li> | + | <li style="font-size: 15px;"> Chitosan-Agarose Hydrogels</li> |
<ul> | <ul> | ||
| − | <li> With new approach: first dissolving of agarose, then adding chitosan and acetic acid.</li> | + | <li style="font-size: 15px;"> With new approach: first dissolving of agarose, then adding chitosan and acetic acid.</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Agarose 1%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Agarose 1%</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Agarose 2%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Agarose 2%</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Agarose 3%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Agarose 3%</li> |
| − | <li> Chitosan 2%, Acetic acid 1%, Agarose 1%</li> | + | <li style="font-size: 15px;"> Chitosan 2%, Acetic acid 1%, Agarose 1%</li> |
| − | <li> Chitosan 2%, Acetic acid 1%, Agarose 2%</li> | + | <li style="font-size: 15px;"> Chitosan 2%, Acetic acid 1%, Agarose 2%</li> |
| − | <li> Chitosan 2%, Acetic acid 1%, Agarose 3%</li> | + | <li style="font-size: 15px;"> Chitosan 2%, Acetic acid 1%, Agarose 3%</li> |
</ul> | </ul> | ||
| − | <li> Chitosan-Gelatine Hydrogels</li> | + | <li style="font-size: 15px;"> Chitosan-Gelatine Hydrogels</li> |
<ul> | <ul> | ||
| − | <li> Different concentration of gelatine were dissolved in water</li> | + | <li style="font-size: 15px;"> Different concentration of gelatine were dissolved in water</li> |
| − | <li> 1 % Acetic acid and 1% chitosan were added</li> | + | <li style="font-size: 15px;"> 1 % Acetic acid and 1% chitosan were added</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Gelatine 1%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Gelatine 1%</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Gelatine 2%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Gelatine 2%</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Gelatine 3%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Gelatine 3%</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,667: | Line 1,667: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Put Succinic Anhydride and Chitosan in acetic acid in a three necked flask</li> | + | <li style="font-size: 15px;"> Put Succinic Anhydride and Chitosan in acetic acid in a three necked flask</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,680: | Line 1,680: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> 3D printer crashed again. Continuing with software and setup design.</li> | + | <li style="font-size: 15px;"> 3D printer crashed again. Continuing with software and setup design.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,698: | Line 1,698: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,712: | Line 1,712: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Evaluation</li> | + | <li style="font-size: 15px;"> Evaluation</li> |
<ul> | <ul> | ||
| − | <li> Plasmid extraction of pSB1C3-NodB-His and pUPD-NodB-His via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction of pSB1C3-NodB-His and pUPD-NodB-His via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
| − | <li> Cloning T7-CODvc in pSB1C3</li> | + | <li style="font-size: 15px;"> Cloning T7-CODvc in pSB1C3</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-T7-CODvc and pSB1C3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-T7-CODvc and pSB1C3 with Xba1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1C3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1C3</li> |
| − | <li> PCR clean-up</li> | + | <li style="font-size: 15px;"> PCR clean-up</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Cloning CODvc in pSB1C3</li> | + | <li style="font-size: 15px;"> Cloning CODvc in pSB1C3</li> |
<ul> | <ul> | ||
| − | <li> Digesting pSB1C3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pSB1C3 with Xba1 and Pst1</li> |
| − | <li> Digesting pUPD-T7-CODvc with Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-T7-CODvc with Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1C3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1C3</li> |
| − | <li> PCR clean-up</li> | + | <li style="font-size: 15px;"> PCR clean-up</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-T7-CODvc and pSB1C3-CODvc into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-T7-CODvc and pSB1C3-CODvc into Top10 via heat-shock</li> |
| − | <li> cPCR of pSB1C3-T7-CODvc and pSB1C3-CODvc</li> | + | <li style="font-size: 15px;"> cPCR of pSB1C3-T7-CODvc and pSB1C3-CODvc</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Over-night cultures of pSB1C3-T7-CODvc and pSB1C3-CODvc</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-T7-CODvc and pSB1C3-CODvc</li> |
| − | <li> Plasmid extraction of pSB1C3-CODvc via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction of pSB1C3-CODvc via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,750: | Line 1,750: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> hydrogel with beta-GP</li> | + | <li style="font-size: 15px;"> hydrogel with beta-GP</li> |
<ul> | <ul> | ||
| − | <li> Hydrogels were produced as in [doi: 10.3390/ijms151017765]</li> | + | <li style="font-size: 15px;"> Hydrogels were produced as in [doi: 10.3390/ijms151017765]</li> |
| − | <li> No solid gel could be archieved, but after autoclaving it showed more stability, but was dropped to to its expensiveness</li> | + | <li style="font-size: 15px;"> No solid gel could be archieved, but after autoclaving it showed more stability, but was dropped to to its expensiveness</li> |
| − | <li> Chitosan 1,8% in 0,2M acetic acid, cooled at 4°C for 20min</li> | + | <li style="font-size: 15px;"> Chitosan 1,8% in 0,2M acetic acid, cooled at 4°C for 20min</li> |
| − | <li> beta-glycerol-phosphate disodium salt 50% w/v was added dropwise, ratio 9:1 - Chitosan:beta-G.P.</li> | + | <li style="font-size: 15px;"> beta-glycerol-phosphate disodium salt 50% w/v was added dropwise, ratio 9:1 - Chitosan:beta-G.P.</li> |
| − | <li> Stirred for 20min and poured</li> | + | <li style="font-size: 15px;"> Stirred for 20min and poured</li> |
<ul> | <ul> | ||
| − | <li> No solid gel</li> | + | <li style="font-size: 15px;"> No solid gel</li> |
</ul> | </ul> | ||
| − | <li> hydrogel basic with less beta-GP</li> | + | <li style="font-size: 15px;"> hydrogel basic with less beta-GP</li> |
| − | <li> 2% chitosan and 1% acetic acid + 8% beta-GP. was placed at 39° C for 15h</li> | + | <li style="font-size: 15px;"> 2% chitosan and 1% acetic acid + 8% beta-GP. was placed at 39° C for 15h</li> |
<ul> | <ul> | ||
| − | <li> no changes, still not solid</li> | + | <li style="font-size: 15px;"> no changes, still not solid</li> |
</ul> | </ul> | ||
| − | <li> Using glutaraldehyde</li> | + | <li style="font-size: 15px;"> Using glutaraldehyde</li> |
| − | <li> 1% chitosan + 1% acetic acid + 1% glutaraldehyde</li> | + | <li style="font-size: 15px;"> 1% chitosan + 1% acetic acid + 1% glutaraldehyde</li> |
| − | <li> 1% chitosan + 1% acetic acid + 3% glutaraldehyde</li> | + | <li style="font-size: 15px;"> 1% chitosan + 1% acetic acid + 3% glutaraldehyde</li> |
| − | <li> 1% chitosan + 1% acetic acid + 5% glutaraldehyde</li> | + | <li style="font-size: 15px;"> 1% chitosan + 1% acetic acid + 5% glutaraldehyde</li> |
| − | <li> 2% chitosan + 1% acetic acid + 10% glutaraldehyde</li> | + | <li style="font-size: 15px;"> 2% chitosan + 1% acetic acid + 10% glutaraldehyde</li> |
<ul> | <ul> | ||
| − | <li> no solid gel</li> | + | <li style="font-size: 15px;"> no solid gel</li> |
</ul> | </ul> | ||
| − | <li> placed at 55°C for 12 and 24 h</li> | + | <li style="font-size: 15px;"> placed at 55°C for 12 and 24 h</li> |
<ul> | <ul> | ||
| − | <li> still not solid</li> | + | <li style="font-size: 15px;"> still not solid</li> |
</ul> | </ul> | ||
| − | <li> determine and pH-level</li> | + | <li style="font-size: 15px;"> determine and pH-level</li> |
| − | <li> 0,2 M chitosan - pH: 4,7</li> | + | <li style="font-size: 15px;"> 0,2 M chitosan - pH: 4,7</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,791: | Line 1,791: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Put Succinic Anhydride and Chitosan in acetic acid in a three necked flask</li> | + | <li style="font-size: 15px;"> Put Succinic Anhydride and Chitosan in acetic acid in a three necked flask</li> |
| − | <li> Centrifugate concentration 1 at 5000 rpm</li> | + | <li style="font-size: 15px;"> Centrifugate concentration 1 at 5000 rpm</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,805: | Line 1,805: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Repairing 3D printer. Continuing with software and setup design.</li> | + | <li style="font-size: 15px;"> Repairing 3D printer. Continuing with software and setup design.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,823: | Line 1,823: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Mutagenese PCR</li> | + | <li style="font-size: 15px;"> Mutagenese PCR</li> |
<ul> | <ul> | ||
| − | <li> PCR with primers for mutagenesis PCR</li> | + | <li style="font-size: 15px;"> PCR with primers for mutagenesis PCR</li> |
| − | <li> Phosphorylation of mutagenesis PCR batch</li> | + | <li style="font-size: 15px;"> Phosphorylation of mutagenesis PCR batch</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
| − | <li> DPN1 digestion</li> | + | <li style="font-size: 15px;"> DPN1 digestion</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li> |
| − | <li> ColonyPCR of pSB1C3-Anderson-chiA</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1C3-Anderson-chiA</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Over-night culture of pSB1C3-Anderson-chiA</li> | + | <li style="font-size: 15px;"> Over-night culture of pSB1C3-Anderson-chiA</li> |
| − | <li> Plasmid extraction via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
| − | <li> The mutation still remains in the construct</li> | + | <li style="font-size: 15px;"> The mutation still remains in the construct</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,851: | Line 1,851: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Preparation</li> | + | <li style="font-size: 15px;"> Preparation</li> |
<ul> | <ul> | ||
| − | <li> Disruption of cells via high pressure</li> | + | <li style="font-size: 15px;"> Disruption of cells via high pressure</li> |
| − | <li> ultracentrifuge to separate cell components</li> | + | <li style="font-size: 15px;"> ultracentrifuge to separate cell components</li> |
</ul> | </ul> | ||
| − | <li> Purification</li> | + | <li style="font-size: 15px;"> Purification</li> |
<ul> | <ul> | ||
| − | <li> Purification via ÄKTA</li> | + | <li style="font-size: 15px;"> Purification via ÄKTA</li> |
</ul> | </ul> | ||
| − | <li> Verification</li> | + | <li style="font-size: 15px;"> Verification</li> |
<ul> | <ul> | ||
| − | <li> A SDS-PAGE of the fractions was performed</li> | + | <li style="font-size: 15px;"> A SDS-PAGE of the fractions was performed</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,875: | Line 1,875: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Transformation</li> | + | <li style="font-size: 15px;"> Transformation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pUPD-T7-COD and pUPD-T7-nodB-His into BL21 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pUPD-T7-COD and pUPD-T7-nodB-His into BL21 via heat-shock</li> |
</ul> | </ul> | ||
| − | <li> Over-night cultures of pUPD-T7-nodB-His, pUPD-T7-COD and empty BL21 cells</li> | + | <li style="font-size: 15px;"> Over-night cultures of pUPD-T7-nodB-His, pUPD-T7-COD and empty BL21 cells</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
<ul> | <ul> | ||
| − | <li> Induction with IPTG at OD=0.6</li> | + | <li style="font-size: 15px;"> Induction with IPTG at OD=0.6</li> |
<ul> | <ul> | ||
| − | <li> Taking sample1 before induction</li> | + | <li style="font-size: 15px;"> Taking sample1 before induction</li> |
| − | <li> Taking sample2 three hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample2 three hours after induction</li> |
| − | <li> Taking sample3 six hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample3 six hours after induction</li> |
| − | <li> Taking sample4 the next day</li> | + | <li style="font-size: 15px;"> Taking sample4 the next day</li> |
</ul> | </ul> | ||
| − | <li> Store samples at -20 degree Celsius</li> | + | <li style="font-size: 15px;"> Store samples at -20 degree Celsius</li> |
</ul> | </ul> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
| − | <li> Expression of pUPD-NodB-His by 16 °C and 37°C</li> | + | <li style="font-size: 15px;"> Expression of pUPD-NodB-His by 16 °C and 37°C</li> |
| − | <li> Sequencing</li> | + | <li style="font-size: 15px;"> Sequencing</li> |
<ul> | <ul> | ||
| − | <li> Plasmid extraction of pUPD-CODvc via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction of pUPD-CODvc via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,908: | Line 1,908: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Chitosan-Gelantine</li> | + | <li style="font-size: 15px;"> Chitosan-Gelantine</li> |
<ul> | <ul> | ||
| − | <li> 2%Chitosan - 0,5 % acetic acid - 2% Gelantine:</li> | + | <li style="font-size: 15px;"> 2%Chitosan - 0,5 % acetic acid - 2% Gelantine:</li> |
| − | <li> 2%Chitosan - 1 % acetic acid - 2% Gelantine:</li> | + | <li style="font-size: 15px;"> 2%Chitosan - 1 % acetic acid - 2% Gelantine:</li> |
| − | <li> 3%Chitosan - 0,5 % acetic acid - 2% Gelantine:</li> | + | <li style="font-size: 15px;"> 3%Chitosan - 0,5 % acetic acid - 2% Gelantine:</li> |
| − | <li> 3%Chitosan - 0,5 % acetic acid - 3% Gelantine:</li> | + | <li style="font-size: 15px;"> 3%Chitosan - 0,5 % acetic acid - 3% Gelantine:</li> |
</ul> | </ul> | ||
| − | <li> DAB-Gel #2</li> | + | <li style="font-size: 15px;"> DAB-Gel #2</li> |
<ul> | <ul> | ||
| − | <li> redone experiments DAB-Gel</li> | + | <li style="font-size: 15px;"> redone experiments DAB-Gel</li> |
| − | <li> same results</li> | + | <li style="font-size: 15px;"> same results</li> |
</ul> | </ul> | ||
| − | <li> centrifugation of chitosan solution</li> | + | <li style="font-size: 15px;"> centrifugation of chitosan solution</li> |
<ul> | <ul> | ||
| − | <li> 2%chitosan + 1% acetic acid</li> | + | <li style="font-size: 15px;"> 2%chitosan + 1% acetic acid</li> |
| − | <li> 5000 rpm for 20 min</li> | + | <li style="font-size: 15px;"> 5000 rpm for 20 min</li> |
| − | <li> no supernatent, visually didnt change anything</li> | + | <li style="font-size: 15px;"> no supernatent, visually didnt change anything</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,937: | Line 1,937: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Put Succinic Anhydride and Chitosan in acetic acid in a three necked flask</li> | + | <li style="font-size: 15px;"> Put Succinic Anhydride and Chitosan in acetic acid in a three necked flask</li> |
| − | <li> Consultaion of Prof. Schoenherr</li> | + | <li style="font-size: 15px;"> Consultaion of Prof. Schoenherr</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,951: | Line 1,951: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> 3D printer is working again. Bearings are excluded due to their degrees of freedom. Idea about replacing the pinhole with a glassfiber comes up, glassfiber is ordered. Discussion about changing the light source start due to the lack of light intensity.</li> | + | <li style="font-size: 15px;"> 3D printer is working again. Bearings are excluded due to their degrees of freedom. Idea about replacing the pinhole with a glassfiber comes up, glassfiber is ordered. Discussion about changing the light source start due to the lack of light intensity.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,969: | Line 1,969: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Mutagenese PCR</li> | + | <li style="font-size: 15px;"> Mutagenese PCR</li> |
<ul> | <ul> | ||
| − | <li> PCR with primers for mutagenesis PCR</li> | + | <li style="font-size: 15px;"> PCR with primers for mutagenesis PCR</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
| − | <li> DPN1 digestion</li> | + | <li style="font-size: 15px;"> DPN1 digestion</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li> |
| − | <li> ColonyPCR of pSB1C3-Anderson-chiA</li> | + | <li style="font-size: 15px;"> ColonyPCR of pSB1C3-Anderson-chiA</li> |
| − | <li> Gelelectrophoresis</li> | + | <li style="font-size: 15px;"> Gelelectrophoresis</li> |
| − | <li> Overnightculture of pSB1C3-Anderson-chiA</li> | + | <li style="font-size: 15px;"> Overnightculture of pSB1C3-Anderson-chiA</li> |
| − | <li> Plasmid extraction via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
| − | <li> The mutation still remains in the construct</li> | + | <li style="font-size: 15px;"> The mutation still remains in the construct</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 1,996: | Line 1,996: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Verification</li> | + | <li style="font-size: 15px;"> Verification</li> |
<ul> | <ul> | ||
| − | <li> Buffer exchange via PD10 columns</li> | + | <li style="font-size: 15px;"> Buffer exchange via PD10 columns</li> |
| − | <li> UPDTM Glycosyltransferase Assay from Promega</li> | + | <li style="font-size: 15px;"> UPDTM Glycosyltransferase Assay from Promega</li> |
</ul> | </ul> | ||
| − | <li> Cloning nodC into pSB1C3</li> | + | <li style="font-size: 15px;"> Cloning nodC into pSB1C3</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-nodC with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-nodC with Xba1 and Pst1</li> |
| − | <li> Digesting pSB1C3 with Xba1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pSB1C3 with Xba1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1C3</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1C3</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Cloning nodC into pSB1C3-AraC</li> | + | <li style="font-size: 15px;"> Cloning nodC into pSB1C3-AraC</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-Anderson-nodC with Nhe1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pUPD-Anderson-nodC with Nhe1 and Pst1</li> |
| − | <li> Digesting pSB1C3-AraC with Spe1 and Pst1</li> | + | <li style="font-size: 15px;"> Digesting pSB1C3-AraC with Spe1 and Pst1</li> |
| − | <li> Dephosphorylating the backbone pSB1C3-AraC</li> | + | <li style="font-size: 15px;"> Dephosphorylating the backbone pSB1C3-AraC</li> |
| − | <li> Ligation with T4 Ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 Ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into Top10 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into Top10 via heat-shock</li> |
| − | <li> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> | + | <li style="font-size: 15px;"> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li> |
| − | <li> Plasmid extraction via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,032: | Line 2,032: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Glycerol stocks</li> | + | <li style="font-size: 15px;"> Glycerol stocks</li> |
<ul> | <ul> | ||
| − | <li> Glycerol stocks of pSB1C3-CODvc were made</li> | + | <li style="font-size: 15px;"> Glycerol stocks of pSB1C3-CODvc were made</li> |
</ul> | </ul> | ||
| − | <li> Transformation</li> | + | <li style="font-size: 15px;"> Transformation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pUPD-T7-COD into Top10 and pUPD-T7-nodB-His into BL21 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pUPD-T7-COD into Top10 and pUPD-T7-nodB-His into BL21 via heat-shock</li> |
</ul> | </ul> | ||
| − | <li> Over-night cultures of pUPD-T7-nodB-His, pUPD-T7-COD and empty BL21 cells</li> | + | <li style="font-size: 15px;"> Over-night cultures of pUPD-T7-nodB-His, pUPD-T7-COD and empty BL21 cells</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
<ul> | <ul> | ||
| − | <li> Induction with IPTG at OD=0.6</li> | + | <li style="font-size: 15px;"> Induction with IPTG at OD=0.6</li> |
<ul> | <ul> | ||
| − | <li> Taking sample1 before induction</li> | + | <li style="font-size: 15px;"> Taking sample1 before induction</li> |
| − | <li> Taking sample2 three hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample2 three hours after induction</li> |
| − | <li> Taking sample3 six hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample3 six hours after induction</li> |
| − | <li> Taking sample4 the next day</li> | + | <li style="font-size: 15px;"> Taking sample4 the next day</li> |
</ul> | </ul> | ||
| − | <li> Store samples at -20 degree Celsius</li> | + | <li style="font-size: 15px;"> Store samples at -20 degree Celsius</li> |
</ul> | </ul> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
| − | <li> Restriction</li> | + | <li style="font-size: 15px;"> Restriction</li> |
<ul> | <ul> | ||
| − | <li> Digesting pUPD-T7-nodB-His with Nhe1-Hf</li> | + | <li style="font-size: 15px;"> Digesting pUPD-T7-nodB-His with Nhe1-Hf</li> |
| − | <li> Gel-filtration</li> | + | <li style="font-size: 15px;"> Gel-filtration</li> |
| − | <li> Ligation with T4 ligase</li> | + | <li style="font-size: 15px;"> Ligation with T4 ligase</li> |
</ul> | </ul> | ||
| − | <li> Transformation and evaluation</li> | + | <li style="font-size: 15px;"> Transformation and evaluation</li> |
<ul> | <ul> | ||
| − | <li> Transforming pUPD-T7-NodB-His into Top10 and BL21 via heat-shock</li> | + | <li style="font-size: 15px;"> Transforming pUPD-T7-NodB-His into Top10 and BL21 via heat-shock</li> |
| − | <li> Plasmid extraction of pUPD-T7-NodB-His and pUPD-T7-NodB-His (dig. with Pst1-Hf) via Mini-prep-kit and sequencing</li> | + | <li style="font-size: 15px;"> Plasmid extraction of pUPD-T7-NodB-His and pUPD-T7-NodB-His (dig. with Pst1-Hf) via Mini-prep-kit and sequencing</li> |
</ul> | </ul> | ||
| − | <li> Over-night cultures of pUPD-T7-nodB-His, pUPD-T7-NodB-His (dig. with Pst1-Hf) and empty BL21 cells</li> | + | <li style="font-size: 15px;"> Over-night cultures of pUPD-T7-nodB-His, pUPD-T7-NodB-His (dig. with Pst1-Hf) and empty BL21 cells</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
<ul> | <ul> | ||
| − | <li> Induction with IPTG at OD=0.6</li> | + | <li style="font-size: 15px;"> Induction with IPTG at OD=0.6</li> |
<ul> | <ul> | ||
| − | <li> Taking sample1 before induction</li> | + | <li style="font-size: 15px;"> Taking sample1 before induction</li> |
| − | <li> Taking sample2 three hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample2 three hours after induction</li> |
| − | <li> Taking sample3 six hours after induction</li> | + | <li style="font-size: 15px;"> Taking sample3 six hours after induction</li> |
| − | <li> Taking sample4 the next day</li> | + | <li style="font-size: 15px;"> Taking sample4 the next day</li> |
</ul> | </ul> | ||
| − | <li> Store samples at -20 degree Celsius</li> | + | <li style="font-size: 15px;"> Store samples at -20 degree Celsius</li> |
</ul> | </ul> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,089: | Line 2,089: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Testing of different gels</li> | + | <li style="font-size: 15px;"> Testing of different gels</li> |
<ul> | <ul> | ||
| − | <li> 1%Chitosan - 1 % acetic acid - 1% Gelantine: good</li> | + | <li style="font-size: 15px;"> 1%Chitosan - 1 % acetic acid - 1% Gelantine: good</li> |
| − | <li> 1,5%Chitosan - 0,5 % acetic acid - 2% Agar:</li> | + | <li style="font-size: 15px;"> 1,5%Chitosan - 0,5 % acetic acid - 2% Agar:</li> |
| − | <li> 0,5%Chitosan - 0,5 % acetic acid - 2% Agar:</li> | + | <li style="font-size: 15px;"> 0,5%Chitosan - 0,5 % acetic acid - 2% Agar:</li> |
| − | <li> 2%Chitosan - 0,5 % acetic acid - 1% Agar:</li> | + | <li style="font-size: 15px;"> 2%Chitosan - 0,5 % acetic acid - 1% Agar:</li> |
| − | <li> 2%Chitosan - 0,5 % acetic acid - 2% Agar:</li> | + | <li style="font-size: 15px;"> 2%Chitosan - 0,5 % acetic acid - 2% Agar:</li> |
| − | <li> 2%Chitosan - 0,5 % acetic acid - 3% Agar:</li> | + | <li style="font-size: 15px;"> 2%Chitosan - 0,5 % acetic acid - 3% Agar:</li> |
| − | <li> 1%Chitosan - 0,5 % acetic acid - 1% Agar:</li> | + | <li style="font-size: 15px;"> 1%Chitosan - 0,5 % acetic acid - 1% Agar:</li> |
| − | <li> 1,5%Chitosan - 0,5 % acetic acid - 2% Agarose: good</li> | + | <li style="font-size: 15px;"> 1,5%Chitosan - 0,5 % acetic acid - 2% Agarose: good</li> |
| − | <li> 0,5%Chitosan - 0,5 % acetic acid - 2% Agarose: too hard</li> | + | <li style="font-size: 15px;"> 0,5%Chitosan - 0,5 % acetic acid - 2% Agarose: too hard</li> |
| − | <li> 1%Chitosan - 1 % acetic acid - 1% Agarose: solid</li> | + | <li style="font-size: 15px;"> 1%Chitosan - 1 % acetic acid - 1% Agarose: solid</li> |
| − | <li> 1%Chitosan - 1 % acetic acid - 2% Agarose: solid</li> | + | <li style="font-size: 15px;"> 1%Chitosan - 1 % acetic acid - 2% Agarose: solid</li> |
| − | <li> 1%Chitosan - 1 % acetic acid - 3% Agarose: not solid</li> | + | <li style="font-size: 15px;"> 1%Chitosan - 1 % acetic acid - 3% Agarose: not solid</li> |
| − | <li> 2%Chitosan - 1 % acetic acid - 1% Agarose: solid</li> | + | <li style="font-size: 15px;"> 2%Chitosan - 1 % acetic acid - 1% Agarose: solid</li> |
| − | <li> 2%Chitosan - 1 % acetic acid - 2% Agarose: not solid</li> | + | <li style="font-size: 15px;"> 2%Chitosan - 1 % acetic acid - 2% Agarose: not solid</li> |
| − | <li> 2%Chitosan - 1 % acetic acid - 3% Agarose: not solid</li> | + | <li style="font-size: 15px;"> 2%Chitosan - 1 % acetic acid - 3% Agarose: not solid</li> |
</ul> | </ul> | ||
| − | <li> Chitosan-Agarose Hydrogel</li> | + | <li style="font-size: 15px;"> Chitosan-Agarose Hydrogel</li> |
<ul> | <ul> | ||
| − | <li> Dissolving of agarose in water,</li> | + | <li style="font-size: 15px;"> Dissolving of agarose in water,</li> |
| − | <li> Simultaneously dissolving of chitosan in 1% acetic acid and stirring.</li> | + | <li style="font-size: 15px;"> Simultaneously dissolving of chitosan in 1% acetic acid and stirring.</li> |
| − | <li> When chitosan is dissolved, mixing of both solutions.</li> | + | <li style="font-size: 15px;"> When chitosan is dissolved, mixing of both solutions.</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Agarose 1%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Agarose 1%</li> |
| − | <li> Chitosan 1%, Acetic acid 1%, Agarose 1%</li> | + | <li style="font-size: 15px;"> Chitosan 1%, Acetic acid 1%, Agarose 1%</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,126: | Line 2,126: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Changed the light source to smartphone LED and LEDs in general. Both deliver enough intensity for the cam and the two micrometres scaled bar becomes visible. Working on a clever manipulator continues. Software make further progress; first pictures are processed.</li> | + | <li style="font-size: 15px;"> Changed the light source to smartphone LED and LEDs in general. Both deliver enough intensity for the cam and the two micrometres scaled bar becomes visible. Working on a clever manipulator continues. Software make further progress; first pictures are processed.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,139: | Line 2,139: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Spin Coat the hydrogels on microscope sildes</li> | + | <li style="font-size: 15px;"> Spin Coat the hydrogels on microscope sildes</li> |
| − | <li> Modificate the surface with either DMSO and succinic anhydride or Aceton and succinic anhydride</li> | + | <li style="font-size: 15px;"> Modificate the surface with either DMSO and succinic anhydride or Aceton and succinic anhydride</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,158: | Line 2,158: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Assay</li> | + | <li style="font-size: 15px;"> Assay</li> |
<ul> | <ul> | ||
| − | <li> Repetition of UDPTM Glycosyltransferase Assay twice</li> | + | <li style="font-size: 15px;"> Repetition of UDPTM Glycosyltransferase Assay twice</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,173: | Line 2,173: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> pUPD-NodB-His via Äkta pure</li> | + | <li style="font-size: 15px;"> pUPD-NodB-His via Äkta pure</li> |
<ul> | <ul> | ||
| − | <li> Over-night cultures of pUPD-T7-nodB-His</li> | + | <li style="font-size: 15px;"> Over-night cultures of pUPD-T7-nodB-His</li> |
| − | <li> Growing a main culture</li> | + | <li style="font-size: 15px;"> Growing a main culture</li> |
| − | <li> Induction with IPTG at OD=0.6</li> | + | <li style="font-size: 15px;"> Induction with IPTG at OD=0.6</li> |
| − | <li> Buffer exchange via PD10 coloumns</li> | + | <li style="font-size: 15px;"> Buffer exchange via PD10 coloumns</li> |
</ul> | </ul> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
<ul> | <ul> | ||
| − | <li> A band is visible by a size of 23kDa, accordingly NodB is present</li> | + | <li style="font-size: 15px;"> A band is visible by a size of 23kDa, accordingly NodB is present</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,195: | Line 2,195: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Gel cintaining quercetin</li> | + | <li style="font-size: 15px;"> Gel cintaining quercetin</li> |
<ul> | <ul> | ||
| − | <li> 2% Chitosan solved with 0,5% acetic acid</li> | + | <li style="font-size: 15px;"> 2% Chitosan solved with 0,5% acetic acid</li> |
| − | <li> beta-glycerol-phosphate disodium salt was added dropwise (0,1g/mL)</li> | + | <li style="font-size: 15px;"> beta-glycerol-phosphate disodium salt was added dropwise (0,1g/mL)</li> |
| − | <li> quercetin 200mg/mL</li> | + | <li style="font-size: 15px;"> quercetin 200mg/mL</li> |
| − | <li> DMSO was added 0,15%/0,2%/0,5%/1%, mixed into the solution</li> | + | <li style="font-size: 15px;"> DMSO was added 0,15%/0,2%/0,5%/1%, mixed into the solution</li> |
| − | <li> not any of the gels were solid enough</li> | + | <li style="font-size: 15px;"> not any of the gels were solid enough</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,214: | Line 2,214: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> A Fresnel lens in the DVD pickup eluded the team with a possible hologram. Expanding the laser diode from the pick-up and trying to couple its light into a glassfiber was unsatisfying due to low transmission. First idea of own micromanipulator is printed, needs improvement. Software is able to fully process holograms. 3D output will be included.</li> | + | <li style="font-size: 15px;"> A Fresnel lens in the DVD pickup eluded the team with a possible hologram. Expanding the laser diode from the pick-up and trying to couple its light into a glassfiber was unsatisfying due to low transmission. First idea of own micromanipulator is printed, needs improvement. Software is able to fully process holograms. 3D output will be included.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,227: | Line 2,227: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Putting the microscope sildes in a EDC/NHS solution</li> | + | <li style="font-size: 15px;"> Putting the microscope sildes in a EDC/NHS solution</li> |
| − | <li> Put it afterwards in the peptide solution</li> | + | <li style="font-size: 15px;"> Put it afterwards in the peptide solution</li> |
| − | <li> Verfication of the concept in protease solution</li> | + | <li style="font-size: 15px;"> Verfication of the concept in protease solution</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,247: | Line 2,247: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Verification</li> | + | <li style="font-size: 15px;"> Verification</li> |
<ul> | <ul> | ||
| − | <li> Repetition of UDPTM Glycosyltransferase Assay</li> | + | <li style="font-size: 15px;"> Repetition of UDPTM Glycosyltransferase Assay</li> |
| − | <li> A thin-layer-chromatography for verification of chitin</li> | + | <li style="font-size: 15px;"> A thin-layer-chromatography for verification of chitin</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,263: | Line 2,263: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> A SDS-Page was performed</li> | + | <li style="font-size: 15px;"> A SDS-Page was performed</li> |
| − | <li> Enzyme reaction</li> | + | <li style="font-size: 15px;"> Enzyme reaction</li> |
<ul> | <ul> | ||
| − | <li> Enzyme reaction with 1mM chitin pentamer, 2,5µM NodB and NH4HCO3</li> | + | <li style="font-size: 15px;"> Enzyme reaction with 1mM chitin pentamer, 2,5µM NodB and NH4HCO3</li> |
</ul> | </ul> | ||
| − | <li> Acetic Acid Assay from Megazym</li> | + | <li style="font-size: 15px;"> Acetic Acid Assay from Megazym</li> |
<ul> | <ul> | ||
| − | <li> The assay was performed two times</li> | + | <li style="font-size: 15px;"> The assay was performed two times</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,283: | Line 2,283: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Chitosan hydrogel solidified in alginate-quercetin</li> | + | <li style="font-size: 15px;"> Chitosan hydrogel solidified in alginate-quercetin</li> |
<ul> | <ul> | ||
| − | <li> different incubation times were tested;</li> | + | <li style="font-size: 15px;"> different incubation times were tested;</li> |
| − | <li> after 12 h: the gel was not solid</li> | + | <li style="font-size: 15px;"> after 12 h: the gel was not solid</li> |
| − | <li> after 18 h: the gel didn't had the required solidarity</li> | + | <li style="font-size: 15px;"> after 18 h: the gel didn't had the required solidarity</li> |
| − | <li> as well as chitosan 1g/2g/3g</li> | + | <li style="font-size: 15px;"> as well as chitosan 1g/2g/3g</li> |
</ul> | </ul> | ||
| − | <li> The most promising gel:</li> | + | <li style="font-size: 15px;"> The most promising gel:</li> |
<ul> | <ul> | ||
| − | <li> 2%chitosan</li> | + | <li style="font-size: 15px;"> 2%chitosan</li> |
| − | <li> 100 % dH2O</li> | + | <li style="font-size: 15px;"> 100 % dH2O</li> |
| − | <li> 6h stirring</li> | + | <li style="font-size: 15px;"> 6h stirring</li> |
| − | <li> 0,5 % acetic acid</li> | + | <li style="font-size: 15px;"> 0,5 % acetic acid</li> |
| − | <li> 12h stirring</li> | + | <li style="font-size: 15px;"> 12h stirring</li> |
| − | <li> 12h rest at 4degree Celsius</li> | + | <li style="font-size: 15px;"> 12h rest at 4degree Celsius</li> |
| − | <li> prepare alginate 2% + quercetin (in dmso) at 1%</li> | + | <li style="font-size: 15px;"> prepare alginate 2% + quercetin (in dmso) at 1%</li> |
| − | <li> pour Chitosan onto frozen Alginate/Quercetin</li> | + | <li style="font-size: 15px;"> pour Chitosan onto frozen Alginate/Quercetin</li> |
| − | <li> add more Alginate/Quercetin</li> | + | <li style="font-size: 15px;"> add more Alginate/Quercetin</li> |
| − | <li> rest at 37°C for 24h</li> | + | <li style="font-size: 15px;"> rest at 37°C for 24h</li> |
| − | <li> it was placed in water and the thicknes was measured as well as the weight</li> | + | <li style="font-size: 15px;"> it was placed in water and the thicknes was measured as well as the weight</li> |
| − | <li> after 12 hours: swelling +200%</li> | + | <li style="font-size: 15px;"> after 12 hours: swelling +200%</li> |
| − | <li> after 24 hours: swelling +600%</li> | + | <li style="font-size: 15px;"> after 24 hours: swelling +600%</li> |
</ul> | </ul> | ||
| − | <li> gel produced for spin-coating (see. chemistry subproject)</li> | + | <li style="font-size: 15px;"> gel produced for spin-coating (see. chemistry subproject)</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,319: | Line 2,319: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Different concentrations of peptide and protease to check detection limit</li> | + | <li style="font-size: 15px;"> Different concentrations of peptide and protease to check detection limit</li> |
| − | <li> Put finished films in protease solution</li> | + | <li style="font-size: 15px;"> Put finished films in protease solution</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,333: | Line 2,333: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> 3D printer crashed again. Another workgroup allows to use theirs. Trying to repair our own. Further work on the setup and the software. More pictures acquired with different smartphones and LEDs.</li> | + | <li style="font-size: 15px;"> 3D printer crashed again. Another workgroup allows to use theirs. Trying to repair our own. Further work on the setup and the software. More pictures acquired with different smartphones and LEDs.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,351: | Line 2,351: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Assay</li> | + | <li style="font-size: 15px;"> Assay</li> |
<ul> | <ul> | ||
| − | <li> Repetition of UDPTM Glycosyltransferase Assay</li> | + | <li style="font-size: 15px;"> Repetition of UDPTM Glycosyltransferase Assay</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,366: | Line 2,366: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Acetic Acid Assay from Megazym</li> | + | <li style="font-size: 15px;"> Acetic Acid Assay from Megazym</li> |
<ul> | <ul> | ||
| − | <li> The assay was performed</li> | + | <li style="font-size: 15px;"> The assay was performed</li> |
</ul> | </ul> | ||
| − | <li> A thin-layer chromatographie was performed</li> | + | <li style="font-size: 15px;"> A thin-layer chromatographie was performed</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,383: | Line 2,383: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Reproduce</li> | + | <li style="font-size: 15px;"> Reproduce</li> |
<ul> | <ul> | ||
| − | <li> we had redone our best hydrogel and produced them in a larger scale</li> | + | <li style="font-size: 15px;"> we had redone our best hydrogel and produced them in a larger scale</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,398: | Line 2,398: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Using the DNA submission kit</li> | + | <li style="font-size: 15px;"> Using the DNA submission kit</li> |
<ul> | <ul> | ||
| − | <li> Dispense each 10ul (25ng/ul) of every part in pSB1C3 into a well</li> | + | <li style="font-size: 15px;"> Dispense each 10ul (25ng/ul) of every part in pSB1C3 into a well</li> |
| − | <li> Dry down the plate in a lab laminar flow hood (over night)</li> | + | <li style="font-size: 15px;"> Dry down the plate in a lab laminar flow hood (over night)</li> |
| − | <li> Label and ship the DNA</li> | + | <li style="font-size: 15px;"> Label and ship the DNA</li> |
</ul> | </ul> | ||
</article> | </article> | ||
| Line 2,415: | Line 2,415: | ||
<p><b>Procedure:</b></p> | <p><b>Procedure:</b></p> | ||
<article> | <article> | ||
| − | <li> Repeat the dilution series</li> | + | <li style="font-size: 15px;"> Repeat the dilution series</li> |
| − | <li> Put Succinic Anhydride and Chitosan in acetic acid in a three necked flask</li> | + | <li style="font-size: 15px;"> Put Succinic Anhydride and Chitosan in acetic acid in a three necked flask</li> |
| − | <li> Add EDC/NHS soluion</li> | + | <li style="font-size: 15px;"> Add EDC/NHS soluion</li> |
| − | <li> Add peptide</li> | + | <li style="font-size: 15px;"> Add peptide</li> |
| − | <li> Spin coat the hydrogel and test with protease</li> | + | <li style="font-size: 15px;"> Spin coat the hydrogel and test with protease</li> |
| − | <li> Different concentrations of peptide and protease to check detection limit.</li> | + | <li style="font-size: 15px;"> Different concentrations of peptide and protease to check detection limit.</li> |
| − | <li> Put finished films in protease solution.</li> | + | <li style="font-size: 15px;"> Put finished films in protease solution.</li> |
</ul> | </ul> | ||
</article> | </article> | ||
Revision as of 11:08, 28 October 2017
ChiTUcare
Notebook and Methods
Notebook
2017-04-03 - 2017-04-09
Technics
Superior experiment: Building a DIHM
Procedure:
2017-04-10 - 2017-04-16
Technics
Superior experiment: Building a DIHM
Procedure:
2017-04-17 - 2017-04-23
Technics
Superior experiment: Building a DIHM
Procedure:
2017-04-24 - 2017-04-30
Generally Labwork
Superior experiment: Ordering sequences
Procedure:
- NodC, NodB, CDApuc and ChiA
Technics
Superior experiment: Building a DIHM
Procedure:
2017-05-01 - 2017-05-07
Generelly Labwork
Superior experiment: Cloning via GoldenBraid assembly
Procedure:
- Cloning NodC, NodB, CDApuc and ChiA into the pUPD vector
Technics
Superior experiment: Building a DIHM
Procedure:
2017-05-08 - 2017-05-14
Technics
Superior experiment: Building a DIHM
Procedure:
2017-05-15 - 2017-05-21
Technics
Superior experiment: Building a DIHM
Procedure:
2017-05-22 - 2017-05-28
Technics
Superior experiment: Building a DIHM
Procedure:
2017-05-29 - 2017-06-04
Chitinase A1
Superior experiment: Cloning chiA into pSB1C3
Procedure:
- Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1C3
- Ligation with T4 Ligase
Chitin Synthase
Superior experiment: Cloning nodC into pSB1K3
Procedure:
- Digesting pUPD-Anderson-nodC and pSB1K3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1K3
- Ligation with T4 Ligase
- Transforming pSB1K3-Anderson-nodC into Top10
- Over-night cultures of pSB1K3-Anderson-nodC
- Plasmid extraction via Mini-prep-kit
Chitin Deacetylases
Superior experiment: Cloning nodB into pSB1K3, transformation into Top10, evaluation
Procedure:
- Digesting pUPD-Anderson-nodB and pSB1K3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1K3
- Ligation with T4 Ligase
- Transforming pSB1K3-Anderson-nodB and pUPD-Anderson-CDApuc into Top10 via heat-shock
- ColonyPCR of pSB1K3-Anderson-nodB and pUPD-Anderson-CDApuc
Technics
Superior experiment: Building a DIHM
Procedure:
2017-06-05 - 2017-06-11
Chitinase A1
Superior experiment: Cloning AraC-chiA into pSB1C3, transformation into Top10, evaluation
Procedure:
- Digesting pUPD-Anderson-chiA with Nhe1 and Pst1, pSB1C3-AraC with Spe1 and Pst1
- Dephosphorylating the backbone pSB1C3-AraC
- Ligation with T4 Ligase
- Transforming pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA into Top10 via heat-shock
- ColonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA
- Gelelectrophoresis
- Over-night cultures of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA
- Plasmid extraction via Mini-prep-kit and sequencing
Chitin Synthase
Superior experiment: Cloning nodC into pSB1C3 and pSB1C3-AraC
Procedure:
- Digesting pUPD-Anderson-nodC and pSB1C3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1K3
- Ligation with T4 Ligase
- Transforming pSB1K3-Anderson-nodC into Top10
- ColonyPCR
- Gelelectrophoresis
- Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
- Plasmid extraction via Mini-prep-kit
Chitin Deacetylases
Superior experiment: Evaluation of transformation, retransformation
Procedure:
- Gelelectrophoresis of colonyPCR product
- Repeating the colonyPCR of pSB1C3-Anderson-nodB and pUPD-Anderson-CDApuc
- Retransformation of pSB1C3-Anderson-nodB and pUPD-Anderson-CDApuc into Top10
- Over-night cultures of pSB1C3-Anderson-nodB
- Plasmid extraction via Mini-prep-kit and sequencing
- No insert was found
Technics
Superior experiment: Building a DIHM
Procedure:
2017-06-12 - 2017-06-18
Chitinase A1
Superior experiment: Cloning chiA into pSB1C3
Procedure:
- Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1C3
- Ligation with T4 Ligase
- Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock
Chitin Synthase
Superior experiment: Sequencing of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
Procedure:
- Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
- Plasmid extraction via Mini-prep-kit and sequencing
- Glycerol stocks were made of positive pSB1C3-Anderson-nodC colonies
- Glycerol stocks were made of positive pSB1C3-AraC-nodC colonies
Chitin Deacetylases
Superior experiment: Cloning nodB in pSB1C3, transformation into Top10
Procedure:
- Digesting pUPD-Anderson-nodB and pSB1C3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1C3
- PCR clean-up to
- Ligation with T4 Ligase
- Transforming pSB1C3-Anderson-nodB into Top10 via heat-shock
- No colony was successful
- Cloning nodB into pSB1K3
- Digesting pUPD-Anderson-nodB and pSB1C3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1C3
- PCR clean-up to
- Ligation with T4 Ligase
- Transforming pSB1C3-Anderson-nodB into Top10 via heat-shock
Technics
Superior experiment: Building a DIHM
Procedure:
2017-06-19 - 2017-06-25
Chitinase A1
Superior experiment: Evaluation of transformation
Procedure:
- ColonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA
- Gelelectrophoresis
- Over-night cultures of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA
- Plasmid extraction via Mini-prep-kit and sequencing
Chitin Deacetylases
Superior experiment: Evaluation of transformation, GoldenBraid assembly and DNA-preparation
Procedure:
- Several colonyPCRs were done
- Several gelelectrophoresis
- They showed that the colonyPCRs did not work properly
- GoldenBrain assembly
- Cloning Anderson-CDApuc in pUPD
- Transforming the pUPD-Anderson-CDApuc into Top10 via heat-shock
- Plasmid extraction of pSB1C3-Anderson-nodB and pSB1K3-Anderson-nodB via Mini-prep Kit
Technics
Superior experiment: Building a DIHM
Procedure:
2017-06-26 - 2017-07-02
Chitinase A1
Superior experiment: Gaining more of the construct pSB1C3-AraC-chiA
Procedure:
- Retransforming pSB1C3-AraC-chiA into Top10 via heat-shock
- ColonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA
- Gelelectrophoresis
- Over-night cultures
- Plasmid extraction via Mini-prep-kit
Chitin Deacetylases
Superior experiment: Sequencing and cloning nodB in pSB1C3 without promotor
Procedure:
- Plasmid extraction of pUPD-Anderson-CDApuc via Mini-prep Kit and sequencing
- Digesting pUPD-Anderson-nodB with Pst1
- PCR clean-up
- Digesting pUPD-Anderson-nodB with Pst1 again
- Ligation of nodB and pSB1C3 with T4 Ligase
Technics
Superior experiment: Building a DIHM
Procedure:
2017-07-03 - 2017-07-09
Chitinase A1
Superior experiment: Transformation of pSB1C3-AraC-chiA into BL21
Procedure:
- Transforming pSB1C3-AraC-chiA into BL21 via heat-shock
- ColonyPCR of pSB1C3-AraC-chiA
- Gelelectrophoresis
Chitin Synthase
Superior experiment: Cloning nodC into pSb1K3, pSB1C3 and pSB1C3-AraC, transformation in Top10
Procedure:
- Digesting pUPD-Anderson-nodC with Xba1 and Pst1
- Digesting pSB1K3 with Xba1 and Pst1
- Gelelectrophoresis and gelextraction
- Ligation with T4 Ligase
- Digesting pUPD-Anderson-nodC with Xba1 and Pst1
- Digesting pSB1C3 with Xba1 and Pst1
- Gelelectrophoresis and gelextraction
- Ligation with T4 Ligase
- Digesting pUPD-Anderson-nodC with Xba1 and Pst1
- Digesting pSb1C3-AraC with Spe1 and Pst1
- Gelelectrophoresis and gelextraction
- Ligation with T4 Ligase
- Transforming pSB1K3-Anderson-nodC, pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into Top10 via heat-shock
Chitin Deacetylases
Superior experiment: Transformation pSB1C3-nodB, evaluation and cloning CDApuc into pSB1C3 and pSB1K3 without promotor
Procedure:
- Transforming the pSB1C3-nodB into Top10 via heat-shock
- ColonyPCR of pSB1C3-nodB
- Over-night cultures of pSB1C3-nodB
- Plasmid extraction of pSB1C3-nodB via Mini-prep-kit and sequencing
- Transforming the pSB1C3-CDApuc, pSB1K3-CDApuc, pSB1C3-Anderson-CDApuc and pSB1K3-Anderson-CDApuc into Top10 via heat-shock
- Plasmid extraction of pSB1C3-CDApuc and pSB1K3-CDApuc via Mini-prep Kit
- Transforming the pSB1C3-nodB into Top10 via heat-shock
Technics
Superior experiment: Building a DIHM
Procedure:
2017-07-10 - 2017-07-16
Chitinase A1
Superior experiment: Detection of Chitinase A1 via SDS-Page
Procedure:
- Induction with arabinose at OD=0.6
- Taking sample1 before induction
- Taking sample2 three hours after induction
- Taking sample3 24 hours after induction
- Store samples at -20 degree Celsius
Chitin Synthase
Superior experiment: Evaluation
Procedure:
- colonyPCR of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
- Gelelectrophoresis
- Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21
- Plasmid extraction via Mini-prep-kit and sequencing
Chitin Deacetylases
Superior experiment: Cloning nodB in pSB1K3
Procedure:
- Digesting pSB1C3-nodB and pSB1K3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1K3
- Ligation with T4 Ligase
- Transforming the pSB1K3-nodB into Top10
Technics
Superior experiment: Building a DIHM
Procedure:
2017-07-17 - 2017-07-23
Chitinase A1
Superior experiment: Detection of Chitinase A1 via SDS-Page
Procedure:
- Induction with arabinose at OD=0.6
- Taking sample1 before induction
- Taking sample2 three hours after induction
- Taking sample3 24 hours after induction
- Store samples at -20 degree Celsius
- A SDS-Page was performed
- Transforming pSB1C3-AraC-chiA into Top10 via heat-shock
- ColonyPCR of pSB1C3-AraC-chiA
- Gelelectrophoresis
Chitin Synthase
Superior experiment: Detection of Chitin-Synthase NodC via SDS-Page
Procedure:
Chitin Deacetylases
Superior experiment: Evaluation
Procedure:
- ColonyPCR of pSB1K3-Anderson-nodB
- Plasmid extraction of pSB1C3-Anderson-CDApuc, pSB1K3-Anderson-CDApuc and pSB1C3-CDApuc via Mini-prep Kit
- Restriction digestion and gelelectrophoresis of pSB1C3-Anderson-CDApuc, pSB1K3-Anderson-CDApuc and pSB1C3-CDApuc
Technics
Superior experiment: Building a DIHM
Procedure:
2017-07-24 - 2017-07-30
Chitinase A1
Superior experiment: Transformation in BL21, detection of Chitinase A1 via SDS-Page
Procedure:
- Transforming pSB1C3-AraC-chiA into BL21 via heat-shock
- ColonyPCR of pSB1C3-AraC-chiA
- Gelelectrophoresis
- Over-night cultures of pSB1C3-AraC-chiA and empty BL21 cells
- Induction with arabinose at OD=0.6
- Taking sample1 before induction
- Taking sample2 three hours after induction
- Taking sample3 24 hours after induction
- Store samples at -20 degree Celsius
- It showed the expected band at 46,5 kDa
Chitin Synthase
Superior experiment: Transformation in BL21, detection of Chitin synthase via SDS-Page
Procedure:
- Induction with arabinose at OD=0.6
- Taking sample1 before induction
- Taking sample2 three hours after induction
- Taking sample3 six hours after induction
- Taking sample4 the next day
- Store samples at -20 degree Celsius
Technics
Superior experiment: Building a DIHM
Procedure:
Chitin Deacetylases
Superior experiment: Evaluation of ColonyPCR of pSB1K3-Anderson-nodB and detection of Chitin Deacetylase NodB via SDS-Page
Procedure:
- Plasmid extraction via Mini-prep kit and sequencing,
- Glycerol stocks of pSB1K3-Anderson-nodB were made
- No induction needed as the Anderson-promotor is constitutive
- Taking sample1 at OD=0.6
- Taking sample2 three hours later
- Taking sample3 six hours later
- Taking sample4 24 hours later
- Store samples at -20 degree Celsius
2017-07-31 - 2017-08-06
Chitinase A1
Superior experiment: Cloning chiA into pSB1C3-Anderson in order to delete the point mutation
Procedure:
- Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1C3
- Ligation with T4 Ligase
- Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock
Chitin Synthase
Superior experiment: Detection of Chitin synthase NodC via SDS-Page
Procedure:
Chitin Deacetylases
Superior experiment: Detection of Chitin Deacetylase NodB via SDS-Page
Procedure:
Technics
Superior experiment: Building a DIHM
Procedure:
2017-08-07 - 2017-08-13
Chitinase A1
Superior experiment: Evaluation of transformation
Procedure:
- ColonyPCR of pSB1C3-Anderson-chiA
- Gelelectrophoresis
- Over-night cultures of pSB1C3-Anderson-chiA
- Plasmid extraction via Mini-prep-kit and sequencing
Chitin Synthase
Superior experiment: Transformation in BL21, detection of Chitin-Synthase via SDS-Page
Procedure:
- Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into BL21 via heat-shock
- Induction with arabinose at OD=0.6
- Taking sample1 before induction
- Taking sample2 three hours after induction
- Taking sample3 six hours after induction
- Taking sample4 the next day
- Store samples at -20 degree Celsius
Chitin Deacetylases
Superior experiment: Detection of Chitin Deacetylase NodB via SDS-Page and transformation into BL21
Procedure:
- Transforming pUPD-nodB into BL21 cells
- The pUPD vector does contain the T7-promotor sequence, which was not used before
- pUPD-T7-nodB is used for detection because the T7-promotor is inducible with IPTG
Technics
Superior experiment: Building a DIHM
Procedure:
2017-08-14 - 2017-08-20
Chitinase A1
Superior experiment: Cloning chiA into pSB1C3-Anderson in order to delete the point mutation
Procedure:
- Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1C3
- Ligation with T4 Ligase
- Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock
- ColonyPCR of pSB1C3-Anderson-chiA
- Gelelectrophoresis
- Over-night cultures of pSB1C3-Anderson-chiA
- Plasmid extraction via Mini-prep-kit and sequencing
Chitin Synthase
Superior experiment: Detection of Chitin synthase via SDS-Page
Procedure:
- Induction with arabinose at OD=0.6
- Taking sample1 before induction
- Taking sample2 three hours after induction
- Taking sample3 six hours after induction
- Taking sample4 the next day
- Store samples at -20 degree Celsius
Chitin Deacetylases
Superior experiment: Detection of Chitin Deacetylase NodB via SDS-Page
Procedure:
- Induction with IPTG at OD=0.6
- Taking sample1 before induction
- Taking sample2 three hours later
- Taking sample3 six hours later
- Taking sample4 24 hours later
- Store samples at -20 degree Celsius
- It showed the expected band at 23 kDa
Technics
Superior experiment: Building a DIHM
Procedure:
2017-08-21 - 2017-08-27
Chitinase A1
Superior experiment: Cloning chiA into pSB1C3-Anderson, transformation in Top10
Procedure:
- Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1C3
- Ligation with T4 Ligase
- Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock
- ColonyPCR of pSB1C3-Anderson-chiA
- Gelelektrophoresis
Chitin Synthase
Superior experiment: Detection of Chitin synthase via SDS-Page
Procedure:
- Induction with arabinose at OD=0.6
- Taking sample1 before induction
- Taking sample2 three hours after induction
- Take sample3 six hours after induction
- Take sample4 the next day
- Store samples at -20 degree Celsius
- Media tested: LB, TB and SOC
- Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
Technics
Superior experiment: Building a DIHM
Procedure:
2017-08-28 - 2017-09-03
Chitinase A1
Superior experiment: Cloning chiA into pSB1C3-Anderson, transformation into Top10, evaluation and sequencing
Procedure:
- Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1
- Gelelektrophoresis and gelextraction
- Ligation with T4 Ligase
- Transforming pSB1C3-Anderson-chiA into Top10
- ColonyPCR
- Gelelectrophoresis
- Over-night cultures of pSB1C3-Anderson-chiA
- Plasmid extraction via Mini-prep-kit and sequencing
Chitin Synthase
Superior experiment: Cell-growth test and SDS-Page
Procedure:
- Media tested: LB, TB and SOC
- Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
- tracing cell-growth over 48 hours
- Induction with arabinose at OD=0.6
- Taking sample1 before induction
- Taking sample2 three hours after induction
- Taking sample3 six hours after induction
- Taking sample4 the next day
- Store samples at -20 degree Celsius
Technics
Superior experiment: Building a DIHM
Procedure:
2017-09-04 - 2017-09-10
Chitin Synthase
Superior experiment: SDS-Page and mutagenesis PCR
Procedure:
- Site-directed-mutagenesis: His-Tag to pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
Chitin Deacetylases
Superior experiment: Cloning via GoldenBraid assembly, transformation and evaluation
Procedure:
- Cloning CODvc into pUPD
- Transforming pUPD-T7-CODvc into Top10 via heat-shock
- Over-night cultures of pUPD-T7-CODvc
- Plasmid extraction via Mini-prep-kit and sequencing
Hydrogels
Superior experiment: How to solve chitosan and first tries to produce a dressing
Procedure:
- Different chitosan/acetic acid concentration were tested
- in 100 % Milli-Q H2O
- 1%Chitosan - 0,5 % acetic acid
- 1%Chitosan - 1 % acetic acid
- 1%Chitosan - 1,5 % acetic acid
- 1%Chitosan - 2 % acetic acid
- 1,5%Chitosan - 0,5 % acetic acid
- 1,5%Chitosan - 1 % acetic acid
- 1,5%Chitosan - 1,5 % acetic acid
- 1,5%Chitosan - 2 % acetic acid
- 2%Chitosan - 0,5 % acetic acid
- 2%Chitosan - 1 % acetic acid
- 2%Chitosan - 1,5 % acetic acid
- 2%Chitosan - 2 % acetic acid
- 3%Chitosan - 0,5 % acetic acid
- 3%Chitosan - 1 % acetic acid
- It is possible to solve chitosan, but it needs time and mechanical stirring, the more acid the visose the solution gets
- Chitosan-Agarose Hydrogels
- Chitosan 1%, Acetic acid 1%, Agarose 1%
- Chitosan 1%, Acetic acid 1%, Agarose 2%
- Chitosan 1%, Acetic acid 1%, Agarose 3%
- Chitosan 2%, Acetic acid 1%, Agarose 1%
- Chitosan 2%, Acetic acid 1%, Agarose 2%
- Chitosan 2%, Acetic acid 1%, Agarose 3%
- Chitosan is slighlty soluble.
- first Agarose was dissolved in water by heating. Then acetic acid and chitosan were added -> better results, better soluble
- Chitosan 1%, Acetic acid 1%, Agarose 1%
- Chitosan 1%, Acetic acid 1%, Agarose 3%
- With 1%: promising
- Chitosan-Agar Hydrogels
- Chitosan 1%, Acetic acid 1%, Agar 1%
- Chitosan 1%, Acetic acid 1%, Agar 2%
- Chitosan 1%, Acetic acid 1%, Agar 3%
Technics
Superior experiment: Building a DIHM
Procedure:
2017-09-11 - 2017-09-17
Chitin Synthase
Superior experiment: Mutagenesis PCR, SDS-Page, retransformation of Site-directed mutagenesis products into BL21
Procedure:
- Phosphorylation of mutagenesis PCR batch
- Ligation with T4 Ligase
- DPN1 digestion
- Gelelectrophoresis
- Transforming pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His into Top10 via heat-shock
- ColonyPCR
- Gelelectrophoresis
- Over-night cultures of pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His
- Plasmid extraction via Mini-prep-kit
- Transforming pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His into BL21 via heat-shock
- colonyPCR of pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His
- Gelelectrophoresis
- Over-night cultures of pSB1C3-Anderson-nodC-His and pSB1C3-AraC-nodC-His
- Plasmid extraction via Mini-prep-kit and sequencing
Chitin Deacetylases
Superior experiment: Mutagenesis PCR, transformation and evaluation
Procedure:
- PCR with His-tag primers for mutagenesis PCR
- Phosphorylation of mutagenesis PCR batch
- Ligation with T4 Ligase
- DPN1 digestion
- Transforming pSB1C3-NodB-His and pUPD-NodB-His into Top10 via heat-shock
- ColonyPCR of
- Gelelectrophoresis
- PCR with His-tag primers for mutagenesis PCR
- Phosphorylation of mutagenesis PCR batch
- Ligation with T4 Ligase
- DPN1 digestion
- Transforming pSB1C3-NodB-His and pUPD-NodB-His into Top10 via heat-shock
Hydrogels
Superior experiment: Testing of different gels
Procedure:
- Hydroxyethylcellulose 2,5
- Glycerol 10%
- Milli-Q H2O 87,5%
- no swelling
- Hydroxyethylcellulose was not "Hydroxyethylcellulose 10000", but with a lower viscosity, so the gel did not form a stable gel
- Order of mixing was determed;
- 1) 2% Chitosan, than 97,5% Milli-Q water, than 0,5% acetic acid
- 2) 2% Chitosan, than 50% Milli-Q water, than 0,5% acetic acid, than 47,5% Milli-Q water
- 3) 2% Chitosan, than 0,5% acetic acid, than 98% Milli-Q water
- 4) 97,5% Milli-Q water mixed with 0,5% acetic acid, than 2% Chitosan
- With new approach: first dissolving of agarose, then adding chitosan and acetic acid.
- Chitosan 1%, Acetic acid 1%, Agarose 1%
- Chitosan 1%, Acetic acid 1%, Agarose 2%
- Chitosan 1%, Acetic acid 1%, Agarose 3%
- Chitosan 2%, Acetic acid 1%, Agarose 1%
- Chitosan 2%, Acetic acid 1%, Agarose 2%
- Chitosan 2%, Acetic acid 1%, Agarose 3%
- Different concentration of gelatine were dissolved in water
- 1 % Acetic acid and 1% chitosan were added
- Chitosan 1%, Acetic acid 1%, Gelatine 1%
- Chitosan 1%, Acetic acid 1%, Gelatine 2%
- Chitosan 1%, Acetic acid 1%, Gelatine 3%
Chemistry
Superior experiment: Preperation of N-Succinic-Chitosan concentration 1 and 2
Procedure:
Technics
Superior experiment: Building a DIHM
Procedure:
2017-09-18 - 2017-09-24
Chitin Synthase
Superior experiment: Preparation of SDS-Page
Procedure:
Chitin Deacetylases
Superior experiment: Cloning T7-CODvc and CODvc in pSB1C3
Procedure:
- Plasmid extraction of pSB1C3-NodB-His and pUPD-NodB-His via Mini-prep-kit and sequencing
- Digesting pUPD-T7-CODvc and pSB1C3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1C3
- PCR clean-up
- Ligation with T4 Ligase
- Digesting pSB1C3 with Xba1 and Pst1
- Digesting pUPD-T7-CODvc with Pst1
- Dephosphorylating the backbone pSB1C3
- PCR clean-up
- Ligation with T4 Ligase
- Transforming pSB1C3-T7-CODvc and pSB1C3-CODvc into Top10 via heat-shock
- cPCR of pSB1C3-T7-CODvc and pSB1C3-CODvc
- Gelelectrophoresis
- Over-night cultures of pSB1C3-T7-CODvc and pSB1C3-CODvc
- Plasmid extraction of pSB1C3-CODvc via Mini-prep-kit and sequencing
Hydrogels
Superior experiment: trying to reproduce papers' hydrogels
Procedure:
- Hydrogels were produced as in [doi: 10.3390/ijms151017765]
- No solid gel could be archieved, but after autoclaving it showed more stability, but was dropped to to its expensiveness
- Chitosan 1,8% in 0,2M acetic acid, cooled at 4°C for 20min
- beta-glycerol-phosphate disodium salt 50% w/v was added dropwise, ratio 9:1 - Chitosan:beta-G.P.
- Stirred for 20min and poured
- No solid gel
- hydrogel basic with less beta-GP
- 2% chitosan and 1% acetic acid + 8% beta-GP. was placed at 39° C for 15h
- no changes, still not solid
- Using glutaraldehyde
- 1% chitosan + 1% acetic acid + 1% glutaraldehyde
- 1% chitosan + 1% acetic acid + 3% glutaraldehyde
- 1% chitosan + 1% acetic acid + 5% glutaraldehyde
- 2% chitosan + 1% acetic acid + 10% glutaraldehyde
- no solid gel
- placed at 55°C for 12 and 24 h
- still not solid
- determine and pH-level
- 0,2 M chitosan - pH: 4,7
Chemistry
Superior experiment: Preperation of N-Succinic-Chitosan concentration 3
Procedure:
Technics
Superior experiment: Building a DIHM
Procedure:
2017-09-25 - 2017-10-01
Chitinase A1
Superior experiment: Mutagenesis PCR, transformation and evaluation via sequencing
Procedure:
- PCR with primers for mutagenesis PCR
- Phosphorylation of mutagenesis PCR batch
- Ligation with T4 Ligase
- DPN1 digestion
- Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock
- ColonyPCR of pSB1C3-Anderson-chiA
- Gelelectrophoresis
- Over-night culture of pSB1C3-Anderson-chiA
- Plasmid extraction via Mini-prep-kit and sequencing
Chitin Synthase
Superior experiment: Protein purification
Procedure:
- Disruption of cells via high pressure
- ultracentrifuge to separate cell components
- Purification via ÄKTA
- A SDS-PAGE of the fractions was performed
Chitin Deacetylases
Superior experiment: Detection of NodB via SDS-Page
Procedure:
- Transforming pUPD-T7-COD and pUPD-T7-nodB-His into BL21 via heat-shock
- Induction with IPTG at OD=0.6
- Taking sample1 before induction
- Taking sample2 three hours after induction
- Taking sample3 six hours after induction
- Taking sample4 the next day
- Store samples at -20 degree Celsius
- Plasmid extraction of pUPD-CODvc via Mini-prep-kit and sequencing
Hydrogels
Superior experiment: gelantine as chross-linking agent
Procedure:
- 2%Chitosan - 0,5 % acetic acid - 2% Gelantine:
- 2%Chitosan - 1 % acetic acid - 2% Gelantine:
- 3%Chitosan - 0,5 % acetic acid - 2% Gelantine:
- 3%Chitosan - 0,5 % acetic acid - 3% Gelantine:
- redone experiments DAB-Gel
- same results
- 2%chitosan + 1% acetic acid
- 5000 rpm for 20 min
- no supernatent, visually didnt change anything
Chemistry
Superior experiment: Preperation of N-Succinic-Chitosan concentration 4
Procedure:
Technics
Superior experiment: Building a DIHM
Procedure:
2017-10-02 - 2017-10-08
Chitinase A1
Superior experiment: Mutagenesis PCR, transformation and evaluation via sequencing
Procedure:
- PCR with primers for mutagenesis PCR
- Ligation with T4 Ligase
- DPN1 digestion
- Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock
- ColonyPCR of pSB1C3-Anderson-chiA
- Gelelectrophoresis
- Overnightculture of pSB1C3-Anderson-chiA
- Plasmid extraction via Mini-prep-kit and sequencing
Chitin Synthase
Superior experiment: Verificate the functionality of NodC
Procedure:
- Buffer exchange via PD10 columns
- UPDTM Glycosyltransferase Assay from Promega
- Digesting pUPD-Anderson-nodC with Xba1 and Pst1
- Digesting pSB1C3 with Xba1 and Pst1
- Dephosphorylating the backbone pSB1C3
- Ligation with T4 Ligase
- Digesting pUPD-Anderson-nodC with Nhe1 and Pst1
- Digesting pSB1C3-AraC with Spe1 and Pst1
- Dephosphorylating the backbone pSB1C3-AraC
- Ligation with T4 Ligase
- Transforming pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into Top10 via heat-shock
- Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC
- Plasmid extraction via Mini-prep-kit and sequencing
Chitin Deacetylases
Superior experiment: SDS-Page, Transformation and evaluation
Procedure:
- Glycerol stocks of pSB1C3-CODvc were made
- Transforming pUPD-T7-COD into Top10 and pUPD-T7-nodB-His into BL21 via heat-shock
- Induction with IPTG at OD=0.6
- Taking sample1 before induction
- Taking sample2 three hours after induction
- Taking sample3 six hours after induction
- Taking sample4 the next day
- Store samples at -20 degree Celsius
- Digesting pUPD-T7-nodB-His with Nhe1-Hf
- Gel-filtration
- Ligation with T4 ligase
- Transforming pUPD-T7-NodB-His into Top10 and BL21 via heat-shock
- Plasmid extraction of pUPD-T7-NodB-His and pUPD-T7-NodB-His (dig. with Pst1-Hf) via Mini-prep-kit and sequencing
- Induction with IPTG at OD=0.6
- Taking sample1 before induction
- Taking sample2 three hours after induction
- Taking sample3 six hours after induction
- Taking sample4 the next day
- Store samples at -20 degree Celsius
Hydrogels
Superior experiment: Switching back to agar/agarose
Procedure:
- 1%Chitosan - 1 % acetic acid - 1% Gelantine: good
- 1,5%Chitosan - 0,5 % acetic acid - 2% Agar:
- 0,5%Chitosan - 0,5 % acetic acid - 2% Agar:
- 2%Chitosan - 0,5 % acetic acid - 1% Agar:
- 2%Chitosan - 0,5 % acetic acid - 2% Agar:
- 2%Chitosan - 0,5 % acetic acid - 3% Agar:
- 1%Chitosan - 0,5 % acetic acid - 1% Agar:
- 1,5%Chitosan - 0,5 % acetic acid - 2% Agarose: good
- 0,5%Chitosan - 0,5 % acetic acid - 2% Agarose: too hard
- 1%Chitosan - 1 % acetic acid - 1% Agarose: solid
- 1%Chitosan - 1 % acetic acid - 2% Agarose: solid
- 1%Chitosan - 1 % acetic acid - 3% Agarose: not solid
- 2%Chitosan - 1 % acetic acid - 1% Agarose: solid
- 2%Chitosan - 1 % acetic acid - 2% Agarose: not solid
- 2%Chitosan - 1 % acetic acid - 3% Agarose: not solid
- Dissolving of agarose in water,
- Simultaneously dissolving of chitosan in 1% acetic acid and stirring.
- When chitosan is dissolved, mixing of both solutions.
- Chitosan 1%, Acetic acid 1%, Agarose 1%
- Chitosan 1%, Acetic acid 1%, Agarose 1%
Technics
Superior experiment: Building a DIHM
Procedure:
Chemistry
Superior experiment: Spin Coating and modification of the films
Procedure:
2017-10-09 - 2017-10-15
Chitin Synthase
Superior experiment: Verificate the functionality of NodC
Procedure:
- Repetition of UDPTM Glycosyltransferase Assay twice
Chitin Deacetylases
Superior experiment: Purification
Procedure:
- Over-night cultures of pUPD-T7-nodB-His
- Growing a main culture
- Induction with IPTG at OD=0.6
- Buffer exchange via PD10 coloumns
- A band is visible by a size of 23kDa, accordingly NodB is present
Hydrogels
Superior experiment: Quercetin as cheap crosslinker
Procedure:
- 2% Chitosan solved with 0,5% acetic acid
- beta-glycerol-phosphate disodium salt was added dropwise (0,1g/mL)
- quercetin 200mg/mL
- DMSO was added 0,15%/0,2%/0,5%/1%, mixed into the solution
- not any of the gels were solid enough
Technics
Superior experiment: Building a DIHM
Procedure:
Chemistry
Superior experiment: Coupling of the peptide (ala-ala-phe-7amc) to the hydrogel layers and prove the concept
Procedure:
2017-10-16 - 2017-10-22
Chitin Synthase
Superior experiment: Verificate the functionality of NodC
Procedure:
- Repetition of UDPTM Glycosyltransferase Assay
- A thin-layer-chromatography for verification of chitin
Chitin Deacetylases
Superior experiment: Acetic Acid Assay
Procedure:
- Enzyme reaction with 1mM chitin pentamer, 2,5µM NodB and NH4HCO3
- The assay was performed two times
Hydrogels
Superior experiment: Swelling chitosan hydrogel testing
Procedure:
- different incubation times were tested;
- after 12 h: the gel was not solid
- after 18 h: the gel didn't had the required solidarity
- as well as chitosan 1g/2g/3g
- 2%chitosan
- 100 % dH2O
- 6h stirring
- 0,5 % acetic acid
- 12h stirring
- 12h rest at 4degree Celsius
- prepare alginate 2% + quercetin (in dmso) at 1%
- pour Chitosan onto frozen Alginate/Quercetin
- add more Alginate/Quercetin
- rest at 37°C for 24h
- it was placed in water and the thicknes was measured as well as the weight
- after 12 hours: swelling +200%
- after 24 hours: swelling +600%
Chemistry
Superior experiment: Making dilution series and test some more hydrogel films with protease
Procedure:
Technics
Superior experiment: Building a DIHM
Procedure:
2017-10-23 - 2017-10-29
Chitin Synthase
Superior experiment: Verificate the functionality of NodC
Procedure:
- Repetition of UDPTM Glycosyltransferase Assay
Chitin Deacetylases
Superior experiment: Acetic Acid Assay and thin-layer chromatographie
Procedure:
- The assay was performed
Hydrogels
Superior experiment: reproducing our most promising hydrogels
Procedure:
- we had redone our best hydrogel and produced them in a larger scale
Generelly labwork
Superior experiment: DNA submission
Procedure:
- Dispense each 10ul (25ng/ul) of every part in pSB1C3 into a well
- Dry down the plate in a lab laminar flow hood (over night)
- Label and ship the DNA
Chemistry
Superior experiment: The first realistic prototype
Procedure:
