background-image: url("https://static.igem.org/mediawiki/2017/9/92/Banner_prov.jpg");

<li><a href="https://2017.igem.org/Team:TU_Darmstadt/project/regulation_system">Regulatory System</a></li>

/*the area of the box on full width*/.labnotebox { width: 100%; margin: auto; background-color: #ffffff;}/*the box itself with the content*/.labnote-content { width: 100%; overflow: auto; margin: auto; padding-bottom: 20px; display: none; background-color: #bea; border-bottom-left-radius: 10px; border-bottom-right-radius: 10px; padding-left: 50px; padding-right: 50px;}/*the clickable title*/.labnote-title { padding-bottom: 5px; padding-left: 50px; padding-right: 50px; padding-top: 5px; text-align: justify; margin-top: 20px; margin: auto; cursor: pointer; text-align: center; background-color: #bda; width: 100%; margin-top: 20px; border-radius: 10px;}/*the date*/.labnote-date { padding: 1px; padding-left: 50px; padding-right: 50px; padding-top: 5px; padding-bottom: 5px; text-align: center; margin: auto; margin-top: 20px; margin-bottom: 20px; background-color: #eeeeee; width: 100%; border-radius: 10px;} .h3{font-size: tiny}

<h3> 2017-04-03  -  2017-04-09 </h3>

<h3> Technics </h3>

<li> First team meeting. Getting to know each other. First discussion about probable projects.</li>

<h3> 2017-04-10  -  2017-04-16 </h3>

<h3> Technics </h3>

<li> Further discussion about possible projects and time management. More research is necessary.</li>

<h3> 2017-04-17  -  2017-04-23 </h3>

<h3> Technics </h3>

<li> Discussions lead to the decision for a microscope. For type and functionality research is planned.</li>

<h3> 2017-04-24  -  2017-04-30 </h3>

<h3> Generally Labwork </h3>

<li> Ordering via IDT</li>

<li>  NodC, NodB, CDApuc and ChiA</li>

<h3> Technics </h3>

<li> Discussion on type of microscope. High resolution wanted with minimum cost. More research is necessary.</li>

<h3> 2017-05-01  -  2017-05-07 </h3>

<h3> Generelly Labwork </h3>

<li> GoldenBraid assembly</li>

<li>  Cloning NodC, NodB, CDApuc and ChiA into the pUPD vector</li>

<h3> Technics </h3>

<li> Decision for a holographic microscope. More research about the exact details and what is needed.</li>

<h3> 2017-05-08  -  2017-05-14 </h3>

<h3> Technics </h3>

<li> Ongoing research</li>

<h3> 2017-05-15  -  2017-05-21 </h3>

<h3> Technics </h3>

<li> First parts like the DVD Pickup are ordered. Ongoing research.</li>

<h3> 2017-05-22  -  2017-05-28 </h3>

<h3> Technics </h3>

<li> Pinhole was sponsored by "Applied semiconductor optics". Ongoing research about detection system and DVD pickup control.</li>

<h3> 2017-05-29  -  2017-06-04 </h3>

<h3> Chitinase A1 </h3>

<li> Cloning chiA into pSB1C3</li>

<li> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li>

<li> Dephosphorylating the backbone pSB1C3</li>

<li> Ligation with T4 Ligase</li>

<h3> Chitin Synthase </h3>

<li> Cloning nodC into pSB1K3</li>

<li> Digesting pUPD-Anderson-nodC and pSB1K3 with Xba1 and Pst1</li>

<li> Dephosphorylating the backbone pSB1K3</li>

<li> Ligation with T4 Ligase</li>

<li> Transformation and evaluation</li>

<li> Transforming pSB1K3-Anderson-nodC into Top10</li>

<li> Over-night cultures of pSB1K3-Anderson-nodC</li>

<li> Plasmid extraction via Mini-prep-kit</li>

<h3> Chitin Deacetylases </h3>

<p><b>Superior experiment:</b> Cloning nodB into pSB1K3, transformation into Top10, evaluation</p>

<li> Cloning nodB into pSB1K3</li>

<li> Digesting pUPD-Anderson-nodB and pSB1K3 with Xba1 and Pst1</li>

<li> Dephosphorylating the backbone pSB1K3</li>

<li> Ligation with T4 Ligase</li>

<li> Transformation and evaluation</li>

<li> 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>

<h3> Technics </h3>

<li> Research about possible software starts as well as about micromanipulators. Furthermore continuing research of the last week.</li>

<h3> 2017-06-05  -  2017-06-11 </h3>

<h3> Chitinase A1 </h3>

<li> Cloning chiA into pSB1C3-AraC</li>

<li> Digesting pUPD-Anderson-chiA with Nhe1 and Pst1, pSB1C3-AraC with Spe1 and Pst1</li>

<li> Dephosphorylating the backbone pSB1C3-AraC</li>

<li> Ligation with T4 Ligase</li>

<li> Transformation and evaluation</li>

<li> 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> Gelelectrophoresis</li>

<li> Over-night cultures of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li>

<li> Plasmid extraction via Mini-prep-kit and sequencing</li>

<h3> Chitin Synthase </h3>

<p><b>Superior experiment:</b> Cloning nodC into pSB1C3 and pSB1C3-AraC</p>

<li> Cloning nodC into pSB1C3-Anderson and pSB1C3-AraC</li>

<li> Digesting pUPD-Anderson-nodC and pSB1C3 with Xba1 and Pst1</li>

<li> Dephosphorylating the backbone pSB1K3</li>

<li> Ligation with T4 Ligase</li>

<li> Transformation and evaluation</li>

<li> Transforming pSB1K3-Anderson-nodC into Top10</li>

<li> ColonyPCR</li>

<li> Gelelectrophoresis</li>

<li> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li>

<li> Plasmid extraction via Mini-prep-kit</li>

<h3> Chitin Deacetylases </h3>

<li> Evaluation</li>

<li> Gelelectrophoresis of colonyPCR product</li>

<li> Repeating the colonyPCR of pSB1C3-Anderson-nodB and pUPD-Anderson-CDApuc</li>

<li> Retransformation</li>

<li> Retransformation of pSB1C3-Anderson-nodB and pUPD-Anderson-CDApuc into Top10</li>

<li> Sequencing</li>

<li> Over-night cultures of pSB1C3-Anderson-nodB</li>

<li> Plasmid extraction via Mini-prep-kit and sequencing</li>

<li> No insert was found</li>

<h3> Technics </h3>

<li> Continuing research of last weeks. First ideas of DVD Pickup control and micromanipulators are acquired.</li>

<h3> 2017-06-12  -  2017-06-18 </h3>

<h3> Chitinase A1 </h3>

<p><b>Superior experiment:</b> Cloning chiA into pSB1C3</p>

<li> Cloning pSB1C3-Anderson-chiA</li>

<li> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li>

<li> Dephosphorylating the backbone pSB1C3</li>

<li> Ligation with T4 Ligase</li>

<li> Transformation</li>

<li> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li>

<h3> Chitin Synthase </h3>

<li> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li>

<li> Plasmid extraction via Mini-prep-kit and sequencing</li>

<li> Glycerol stocks of pSB1C3-Anderson-nodC</li>

<li> Glycerol stocks were made of positive pSB1C3-Anderson-nodC colonies</li>

<li> Glycerol stocks of pSB1C3-AraC-nodC</li>

<li> Glycerol stocks were made of positive pSB1C3-AraC-nodC colonies</li>

<h3> Chitin Deacetylases </h3>

<p><b>Superior experiment:</b> Cloning nodB in pSB1C3, transformation into Top10</p>

<li> Cloning nodB into pSB1K3</li>

<li> Digesting pUPD-Anderson-nodB and pSB1C3 with Xba1 and Pst1</li>

<li> Dephosphorylating the backbone pSB1C3</li>

<li> PCR clean-up to</li>

<li> Ligation with T4 Ligase</li>

<li> Transformation and evaluation</li>

<li> Transforming pSB1C3-Anderson-nodB into Top10 via heat-shock</li>

<li> No colony was successful</li>

<li> Cloning nodB into pSB1K3</li>

<li> Digesting pUPD-Anderson-nodB and pSB1C3 with Xba1 and Pst1</li>

<li> Dephosphorylating the backbone pSB1C3</li>

<li> PCR clean-up to</li>

<li> Ligation with T4 Ligase</li>

<li> Transformation and evaluation</li>

<li> Transforming pSB1C3-Anderson-nodB into Top10 via heat-shock</li>

<h3> Technics </h3>

<li> Start working with the 3D printer. Ongoing research.</li>

<h3> 2017-06-19  -  2017-06-25 </h3>

<h3> Chitinase A1 </h3>

<li> Evaluation</li>

<li> ColonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li>

<li> Gelelectrophoresis</li>

<li> Over-night cultures of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li>

<li> Plasmid extraction via Mini-prep-kit and sequencing</li>

<h3> Chitin Deacetylases </h3>

<li> Evaluation</li>

<li> Several colonyPCRs were done</li>

<li> Several gelelectrophoresis</li>

<li> They showed that the colonyPCRs did not work properly</li>

<li> GoldenBrain assembly</li>

<li> Cloning Anderson-CDApuc in pUPD</li>

<li> Transformation</li>

<li> Transforming the pUPD-Anderson-CDApuc into Top10 via heat-shock</li>

<li> Over-night cultures of pSB1C3-Anderson-nodB and pSB1K3-Anderson-nodB</li>

<li> DNA-preparation</li>

<li> Plasmid extraction of pSB1C3-Anderson-nodB and pSB1K3-Anderson-nodB via Mini-prep Kit</li>

<h3> Technics </h3>

<li> 3D printer crashed and needs repair. Ongoing research.</li>

<h3> 2017-06-26  -  2017-07-02 </h3>

<h3> Chitinase A1 </h3>

<li> Retransformation and evaluation</li>

<li> Retransforming pSB1C3-AraC-chiA into Top10 via heat-shock</li>

<li> ColonyPCR of pSB1C3-Anderson-chiA and pSB1C3-AraC-chiA</li>

<li> Gelelectrophoresis</li>

<li> Over-night cultures</li>

<li> Plasmid extraction via Mini-prep-kit</li>

<li> Detection of a point mutation in pSB1C3-Anderson-ChiA</li>

<h3> Chitin Deacetylases </h3>

<p><b>Superior experiment:</b> Sequencing and cloning nodB in pSB1C3 without promotor</p>

<li> DNA-preparation</li>

<li> Plasmid extraction of pUPD-Anderson-CDApuc via Mini-prep Kit and sequencing</li>

<li> Cloning</li>

<li> Digesting pUPD-Anderson-nodB with Pst1</li>

<li> PCR clean-up</li>

<li> Digesting pUPD-Anderson-nodB with Pst1 again</li>

<li> Ligation of nodB and pSB1C3 with T4 Ligase</li>

<h3> Technics </h3>

<li> Still fixing 3D printer. Ongoing research.</li>

<h3> 2017-07-03  -  2017-07-09 </h3>

<h3> Chitinase A1 </h3>

<li> Transformation and evaluation</li>

<li> Transforming pSB1C3-AraC-chiA into BL21 via heat-shock</li>

<li> ColonyPCR of pSB1C3-AraC-chiA</li>

<li> Gelelectrophoresis</li>

<h3> Chitin Synthase </h3>

<li> Cloning nodC into pSB1K3</li>

<li> Digesting pUPD-Anderson-nodC with Xba1 and Pst1</li>

<li> Digesting pSB1K3 with Xba1 and Pst1</li>

<li> Gelelectrophoresis and gelextraction</li>

<li> Ligation with T4 Ligase</li>

<li> Cloning nodC into pSB1C3</li>

<li> Digesting pUPD-Anderson-nodC with Xba1 and Pst1</li>

<li> Digesting pSB1C3 with Xba1 and Pst1</li>

<li> Gelelectrophoresis and gelextraction</li>

<li> Ligation with T4 Ligase</li>

<li> Cloning nodC into pSB1C3-AraC</li>

<li> Digesting pUPD-Anderson-nodC with Xba1 and Pst1</li>

<li> Digesting pSb1C3-AraC with Spe1 and Pst1</li>

<li> Gelelectrophoresis and gelextraction</li>

<li> Ligation with T4 Ligase</li>

<li> Transformation</li>

<li> Transforming pSB1K3-Anderson-nodC, pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC into Top10 via heat-shock</li>

<h3> Chitin Deacetylases </h3>

<p><b>Superior experiment:</b> Transformation pSB1C3-nodB, evaluation and cloning CDApuc into pSB1C3 and pSB1K3 without promotor</p>

<li> Transformation</li>

<li> Transforming the pSB1C3-nodB into Top10 via heat-shock</li>

<li> Evaluation</li>

<li> ColonyPCR of pSB1C3-nodB</li>

<li> Over-night cultures of pSB1C3-nodB</li>

<li> Plasmid extraction of pSB1C3-nodB via Mini-prep-kit and sequencing</li>

<li> Cloning CDApuc into pSB1C3 and pSB1K3 with and without Anderson-promotor</li>

<li> Transformation</li>

<li> Transforming the pSB1C3-CDApuc, pSB1K3-CDApuc, pSB1C3-Anderson-CDApuc and pSB1K3-Anderson-CDApuc into Top10 via heat-shock</li>

<li> Over-night cultures of pSB1C3-CDApuc, pSB1K3-CDApuc, pSB1C3-Anderson-CDApuc and pSB1K3-Anderson-CDApuc</li>

<li> DNA-preparation</li>

<li> Plasmid extraction of pSB1C3-CDApuc and pSB1K3-CDApuc via Mini-prep Kit</li>

<li> Gelelectrophoresis of pSB1C3-CDApuc and pSB1K3-CDApuc</li>

<li> Transformation (repeat)</li>

<li> Transforming the pSB1C3-nodB into Top10 via heat-shock</li>

<h3> Technics </h3>

<li> Learning about CAD and working on manipulators. Continuing last week?s work.</li>

<h3> 2017-07-10  -  2017-07-16 </h3>

<h3> Chitinase A1 </h3>

<li> Over-night cultures of pSB1C3-AraC-chiA and empty BL21 cells</li>

<li> Growing a main culture</li>

<li> Induction with arabinose at OD=0.6</li>

<li> Taking sample1 before induction</li>

<li> Taking sample2 three hours after induction</li>

<li> Taking sample3 24 hours after induction</li>

<li> Store samples at -20 degree Celsius</li>

<h3> Chitin Synthase </h3>

<li> Evaluation of transformations</li>

<li> colonyPCR of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li>

<li> Gelelectrophoresis</li>

<li> Over-night cultures of pSB1C3-Anderson-nodC, pSB1C3-AraC-nodC and empty BL21</li>

<li> Plasmid extraction via Mini-prep-kit and sequencing</li>

<h3> Chitin Deacetylases </h3>

<p><b>Superior experiment:</b> Cloning nodB in pSB1K3</p>

<li> Cloning nodB in pSB1K3</li>

<li> Digesting pSB1C3-nodB and pSB1K3 with Xba1 and Pst1</li>

<li> Dephosphorylating the backbone pSB1K3</li>

<li> Ligation with T4 Ligase</li>

<li> Transformation</li>

<li> Transforming the pSB1K3-nodB into Top10</li>

<h3> Technics </h3>

<li> Continuing last week's work</li>

<h3> 2017-07-17  -  2017-07-23 </h3>

<h3> Chitinase A1 </h3>

<li> Over-night cultures of pSB1C3-AraC-chiA and empty BL21 cells</li>

<li> Growing a main culture</li>

<li> Induction with arabinose at OD=0.6</li>

<li> Taking sample1 before induction</li>

<li> Taking sample2 three hours after induction</li>

<li> Taking sample3 24 hours after induction</li>

<li> Store samples at -20 degree Celsius</li>

<li> SDS-Page</li>

<li> A SDS-Page was performed</li>

<li> Transformation and evaluation</li>

<li> Transforming pSB1C3-AraC-chiA into Top10 via heat-shock</li>

<li> ColonyPCR of pSB1C3-AraC-chiA</li>

<li> Gelelectrophoresis</li>

<h3> Chitin Synthase </h3>

<li> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li>

<li> Growing a main culture</li>

<h3> Chitin Deacetylases </h3>

<li> Evaluation</li>

<li> 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> Restriction digestion and gelelectrophoresis of pSB1C3-Anderson-CDApuc, pSB1K3-Anderson-CDApuc and pSB1C3-CDApuc</li>

<h3> Technics </h3>

<li> Continuing last week's work</li>

<h3> 2017-07-24  -  2017-07-30 </h3>

<h3> Chitinase A1 </h3>

<li> Transformation and evaluation</li>

<li> Transforming pSB1C3-AraC-chiA into BL21 via heat-shock</li>

<li> ColonyPCR of pSB1C3-AraC-chiA</li>

<li> Gelelectrophoresis</li>

<li> Over-night cultures of pSB1C3-AraC-chiA and empty BL21 cells</li>

<li> Growing a main culture</li>

<li> Induction with arabinose at OD=0.6</li>

<li> Taking sample1 before induction</li>

<li> Taking sample2 three hours after induction</li>

<li> Taking sample3 24 hours after induction</li>

<li> Store samples at -20 degree Celsius</li>

<li> A SDS-Page was performed</li>

<li> It showed the expected band at 46,5 kDa</li>

<h3> Chitin Synthase </h3>

<li> 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> Growing a main culture</li>

<li> Induction with arabinose at OD=0.6</li>

<li> Taking sample1 before induction</li>

<li> Taking sample2 three hours after induction</li>

<li> Taking sample3 six hours after induction</li>

<li> Taking sample4 the next day</li>

<li> Store samples at -20 degree Celsius</li>

<li> A SDS-Page was performed</li>

<h3> Technics </h3>

<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>

<h3> Chitin Deacetylases </h3>

<li> After consultation with an expert the CDApuc was deemed incompatible with the project</li>

<li> Evaluation</li>

<li> Plasmid extraction via Mini-prep kit and sequencing,</li>

<li> Glycerol stocks</li>

<li> Glycerol stocks of pSB1K3-Anderson-nodB were made</li>

<li> Over-night cultures of pSB1C3-Anderson-nodB, pSB1C3-Anderson-nodB and empty Top10</li>

<li> Growing a main culture</li>

<li> No induction needed as the Anderson-promotor is constitutive</li>

<li> Taking sample1 at OD=0.6</li>

<li> Taking sample2 three hours later</li>

<li> Taking sample3 six hours later</li>

<li> Taking sample4 24 hours later</li>

<li> Store samples at -20 degree Celsius</li>

<h3> 2017-07-31  -  2017-08-06 </h3>

<h3> Chitinase A1 </h3>

<li> Cloning chiA into pSB1C3</li>

<li> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li>

<li> Dephosphorylating the backbone pSB1C3</li>

<li> Ligation with T4 Ligase</li>

<li> Transformation</li>

<li> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li>

<h3> Chitin Synthase </h3>

<li> Over-night cultures of pSB1C3-Anderson-nodC and pSB1C3-AraC-nodC</li>

<li> Growing a main culture</li>

<h3> Chitin Deacetylases </h3>

<li> A SDS-Page was performed</li>

<h3> Technics </h3>

<li> Pi Cam is ordered. Learning PCB boards. Printing parts for adjustment of pinhole and DVD pickup. Ongoing research about software.</li>

<h3> 2017-08-07  -  2017-08-13 </h3>

<h3> Chitinase A1 </h3>

<li> Evaluation of transformation</li>

<li> ColonyPCR of pSB1C3-Anderson-chiA</li>

<li> Gelelectrophoresis</li>

<li> Over-night cultures of pSB1C3-Anderson-chiA</li>

<li> Plasmid extraction via Mini-prep-kit and sequencing</li>

<li> The mutation still remains in the construct</li>

<h3> Chitin Synthase </h3>

<li> Transformation</li>

<li> 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> Growing a main culture</li>

<li> Induction with arabinose at OD=0.6</li>

<li> Taking sample1 before induction</li>

<li> Taking sample2 three hours after induction</li>

<li> Taking sample3 six hours after induction</li>

<li> Taking sample4 the next day</li>

<li> Store samples at -20 degree Celsius</li>

<li> A SDS-Page was performed</li>

<h3> Chitin Deacetylases </h3>

<li> A SDS-Page was performed</li>

<li> Transformation</li>

<li> Transforming pUPD-nodB into BL21 cells</li>

<li> The pUPD vector does contain the T7-promotor sequence, which was not used before</li>

<li> pUPD-T7-nodB is used for detection because the T7-promotor is inducible with IPTG</li>

<h3> Technics </h3>

<li> First try of acquiring holograms. Probably intensity to low, further improvement necessary.</li>

<h3> 2017-08-14  -  2017-08-20 </h3>

<h3> Chitinase A1 </h3>

<li> Cloning Anderson-chiA into pSB1C3</li>

<li> Digesting pUPD-Anderson-chiA and pSB1C3 with Xba1 and Pst1</li>

<li> Dephosphorylating the backbone pSB1C3</li>

<li> Ligation with T4 Ligase</li>

<li> Transformation and evaluation</li>

<li> Transforming pSB1C3-Anderson-chiA into Top10 via heat-shock</li>

<li> ColonyPCR of pSB1C3-Anderson-chiA</li>

<li> Gelelectrophoresis</li>

<li> Over-night cultures of pSB1C3-Anderson-chiA</li>

<li> Plasmid extraction via Mini-prep-kit and sequencing</li>

<li> The mutation still remains in the construct</li>

<h3> Chitin Synthase </h3>