Difference between revisions of "Team:Lund/Notebook"

 
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       <article class="card">
 
       <article class="card">
 
         <p class="date">Week 26 - 2017/06/28 - 2017/06/30</p>
 
         <p class="date">Week 26 - 2017/06/28 - 2017/06/30</p>
−
        <h5>Obtaining the pETDuet-1 plasmid backbone</h5>
 
 
         <ul>
 
         <ul>
−
           <li>Cell cultivation* (<em>E. coli</em> BL21_DE3) with PETDuet-1 plasmid </li>
+
          <li>Obtaining the pETDuet-1 plasmid backbone</li>
−
           <li>Plasmid isolation* (pETDuet-1) using GeneJET Plasmid Miniprep Kit </li>
+
           <li>Cell cultivation (<em>E. coli</em> BL21-DE3) with pETDuet-1 plasmid </li>
 +
           <li>Plasmid isolation (pETDuet-1) using GeneJET Plasmid Miniprep Kit </li>
 
           <li>Spectrophotometric analysis of plasmids</li>
 
           <li>Spectrophotometric analysis of plasmids</li>
 
           <li>Too low plasmid concentration, therefore we ordered commercial plasmid backbones</li>
 
           <li>Too low plasmid concentration, therefore we ordered commercial plasmid backbones</li>
Line 34: Line 34:
 
         <p class="date">Week 27 (2017/07/03 - 2017/07/07)</p>
 
         <p class="date">Week 27 (2017/07/03 - 2017/07/07)</p>
 
         <ul>
 
         <ul>
−
           <li>Cell cultivation* (<em>E. coli</em> BL21 and <em>E. coli</em> TG1)</li>
+
           <li>Cell cultivation (<em>E. coli</em> BL21 and <em>E. coli</em> TG1)</li>
−
           <li>Preparation of competent cells* using the Inoue Method for both <em>E. coli </em>strains</li>
+
           <li>Preparation of competent cells using the Inoue Method for both <em>E. coli </em>strains</li>
 
           <li>Freezing of competent cells</li>
 
           <li>Freezing of competent cells</li>
 
         </ul>
 
         </ul>
Line 44: Line 44:
 
         <h5>InterLab attempt 1 </h5>
 
         <h5>InterLab attempt 1 </h5>
 
         <ul>
 
         <ul>
−
           <li>Transformation of competent (<em>E. coli</em> DH5ɑ)* cells</li>
+
           <li>Transformation of competent (<em>E. coli</em> DH5ɑ) cells</li>
 
           <li>The transformation did not succeed </li>
 
           <li>The transformation did not succeed </li>
−
           <li>Competent cell test (<em>E. coli</em> TG1 and BL21_DE3) using Competent Cell Test Kit*</li>
+
           <li>Competent cell test (<em>E. coli</em> TG1 and BL21) using Competent Cell Test Kit</li>
 
           <li>Successful competent TG1 and BL21 <em>E. coli</em> cells</li>
 
           <li>Successful competent TG1 and BL21 <em>E. coli</em> cells</li>
 
           <li>Have not calculated the cell efficiency, as cell competency test was only a test experiment </li>
 
           <li>Have not calculated the cell efficiency, as cell competency test was only a test experiment </li>
Line 90: Line 90:
 
         <p class="date">Week 32 (2017/08/07 - 2017/08/11)</p>
 
         <p class="date">Week 32 (2017/08/07 - 2017/08/11)</p>
 
         <ul>
 
         <ul>
−
           <li>The second batch of pETDuet-1 plasmids has arrived </li>
+
           <li>The second batch of pETDuet-1 plasmids arrived </li>
 
           <li>Received DH5ɑ cells from iGEM team Chalmers Gothenburg</li>
 
           <li>Received DH5ɑ cells from iGEM team Chalmers Gothenburg</li>
 
           <li>Making DH5ɑ competent cells</li>
 
           <li>Making DH5ɑ competent cells</li>
Line 132: Line 132:
 
           <li>Inoculation  of the media with both red and white cells growing after transformation to get more DNA to send for sequencing</li>
 
           <li>Inoculation  of the media with both red and white cells growing after transformation to get more DNA to send for sequencing</li>
 
           <li>Plasmid isolation and freezing of DNA</li>
 
           <li>Plasmid isolation and freezing of DNA</li>
−
           <li>repeating InterLab with cells from iGEM DTU Denmark</li>
+
           <li>Repeating InterLab with cells from iGEM DTU Denmark</li>
 
           <li>InterLab measurements resulted in negative values</li>
 
           <li>InterLab measurements resulted in negative values</li>
−
           <li>repeated the plating of the cells by concentrating already grown bacterial culture</li>
+
           <li>Repeated the plating of the cells by concentrating already grown bacterial culture</li>
−
           <li>after 26 h incubation no cells grew  </li>
+
           <li>After 26 h incubation no cells grew  </li>
 
         </ul>
 
         </ul>
 
       </article>
 
       </article>
Line 142: Line 142:
 
         <p class="date">Week 37 (2017/09/11 - 2017/09/17)</p>
 
         <p class="date">Week 37 (2017/09/11 - 2017/09/17)</p>
 
         <ul>
 
         <ul>
−
           <li>digestion and ligation of gBlocks GFP1-9 and gBlock GFP1-9 device</li>
+
           <li>Digestion and ligation of gBlocks GFP1-9 and gBlock GFP1-9 device</li>
−
           <li>agarose gel electrophoresis to see how successful the ligation was</li>
+
           <li>Agarose gel electrophoresis to see how successful the ligation was</li>
−
           <li>transformation of the ligated gBlocks to<em> E. coli</em> BL21</li>
+
           <li>Transformation of the ligated gBlocks to<em> E. coli</em> BL21</li>
−
           <li>transformation of <em>E. coli</em> DH5ɑ cells with the new batch of devices </li>
+
           <li>Transformation of <em>E. coli</em> DH5ɑ cells with the new batch of devices </li>
−
           <li>plating of the transformed cells</li>
+
           <li>Plating of the transformed cells</li>
−
           <li>preparing 10 mL culture media</li>
+
           <li>Preparing 10 mL culture media</li>
 
         </ul>
 
         </ul>
 
       </article>
 
       </article>
Line 154: Line 154:
 
         <p class="date">Week 38 (2017/09/18 - 2017/09/25)</p>
 
         <p class="date">Week 38 (2017/09/18 - 2017/09/25)</p>
 
         <ul>
 
         <ul>
−
           <li>inoculation of 100 mL media with the overnight culture </li>
+
           <li>Inoculation of 100 mL media with the overnight culture </li>
 
           <li>InterLab measurements</li>
 
           <li>InterLab measurements</li>
−
           <li>digestion and ligation of the GFP1-9 device and GFP1-9; ligation was done overnight </li>
+
           <li>Digestion and ligation of the GFP1-9 device and GFP1-9; ligation was done overnight </li>
−
           <li>digestion and ligation of devices for interlab; ligation was done overnight</li>
+
           <li>Digestion and ligation of devices for interlab; ligation was done overnight</li>
−
           <li>colony PCR of InterLab devices to check if the colonies appearing white on the media had GFP inserted</li>
+
           <li>Colony PCR of InterLab devices to check if the colonies appearing white on the media had GFP inserted</li>
 
           <li>PCR products were run on agarose gel</li>
 
           <li>PCR products were run on agarose gel</li>
 
         </ul>
 
         </ul>
Line 173: Line 173:
 
         <ul>
 
         <ul>
 
           <li>InterLab measurements</li>
 
           <li>InterLab measurements</li>
−
           <li>preparing glycerol stocks of <em>E. coli</em> BL21 cells with inserted GFP1-9 and GFP1-9 device</li>
+
           <li>Preparing glycerol stocks of <em>E. coli</em> BL21 cells with inserted GFP1-9 and GFP1-9 device</li>
−
           <li>plating of cells from the glycerol stocks to check if they were prepared correctly</li>
+
           <li>Plating of cells from the glycerol stocks to check if they were prepared correctly</li>
−
           <li>digestion and ligation of the NahR BioBrick</li>
+
           <li>Digestion and ligation of the NahR BioBrick</li>
−
           <li>transformation of NahR BioBrick to <em>E. coli</em> BL21</li>
+
           <li>Transformation of NahR BioBrick to <em>E. coli</em> BL21</li>
−
           <li>colony PCR of the transformed NahR BioBrick</li>
+
           <li>Colony PCR of the transformed NahR BioBrick</li>
−
           <li>plasmid purification of GFP1-9 and GFP1-9 device BioBricks</li>
+
           <li>Plasmid purification of GFP1-9 and GFP1-9 device BioBricks</li>
 
         </ul>
 
         </ul>
 
       </article>
 
       </article>
Line 185: Line 185:
 
         <p class="date">Week 40 (2017/10/02 - 2017/10/08)</p>
 
         <p class="date">Week 40 (2017/10/02 - 2017/10/08)</p>
 
         <ul>
 
         <ul>
−
           <li>harvesting the cells cultivated from the glycerol stock</li>
+
           <li>Harvesting the cells cultivated from the glycerol stock</li>
−
           <li>sonication of cells with the GFP1-9 device insert</li>
+
           <li>Sonication of cells with the GFP1-9 device insert</li>
−
           <li>plating cells with GFP1-9 device insert to measure any possible background fluorescence</li>
+
           <li>Plating cells with GFP1-9 device insert to measure any possible background fluorescence</li>
−
           <li>running SDS-PAGE to check for expression of GFP1-9</li>
+
           <li>Running SDS-PAGE to check for expression of GFP1-9</li>
−
           <li>digestion and ligation of NahR-GFP1-9 device and NahR-sfGFP device</li>
+
           <li>Digestion and ligation of NahR-GFP1-9 device and NahR-sfGFP device</li>
−
           <li>transformation of ligated NahR-GFP1-9 device and NahR-sfGFP device</li>
+
           <li>Transformation of ligated NahR-GFP1-9 device and NahR-sfGFP device</li>
−
           <li>transformation of NahR in pETDuet vector to <em>E. coli</em> BL21, plating of the cells - no growth</li>
+
           <li>Transformation of NahR in pETDuet vector to <em>E. coli</em> BL21, plating of the cells - no growth</li>
−
           <li>agarose gel electrophoresis to check if the ligation succeeded - it did not</li>
+
           <li>Agarose gel electrophoresis to check if the ligation succeeded - it did not</li>
−
           <li>repeated digestion and ligation of NahR-GFP1-9 device and NahR-sfGFP device</li>
+
           <li>Repeated digestion and ligation of NahR-GFP1-9 device and NahR-sfGFP device</li>
−
           <li>repeated transformation of ligated NahR-GFP1-9 device and NahR-sfGFP device</li>
+
           <li>Repeated transformation of ligated NahR-GFP1-9 device and NahR-sfGFP device</li>
 
         </ul>
 
         </ul>
 
       </article>
 
       </article>
Line 201: Line 201:
 
         <p class="date">Week 41 (2017/10/09 - 2017/10/15)</p>
 
         <p class="date">Week 41 (2017/10/09 - 2017/10/15)</p>
 
         <ul>
 
         <ul>
−
           <li>colony PCR of the plated GFP1-9 device</li>
+
           <li>Colony PCR of the plated GFP1-9 device</li>
−
           <li>plating of competent <em>E. coli</em> BL21 cells and <em>E. coli</em> BL21 cells with GFP1-9 insert from the glycerol stock</li>
+
           <li>Plating of competent <em>E. coli</em> BL21 cells and <em>E. coli</em> BL21 cells with GFP1-9 insert from the glycerol stock</li>
−
           <li>digestion and ligation of ER-ɑ device, NahR-GFP1-9  device and NahR-sfGFP device</li>
+
           <li>Digestion and ligation of ER-ɑ device, NahR-GFP1-9  device and NahR-sfGFP device</li>
−
           <li>agarose gel electrophoresis of digested ER-ɑ device, NahR-GFP1-9  device and NahR-sfGFP device products</li>
+
           <li>Agarose gel electrophoresis of digested ER-ɑ device, NahR-GFP1-9  device and NahR-sfGFP device products</li>
−
           <li>transformation of chromoprotein BioBrick (Bba_K1033910) using <em>E. coli</em> BL21 and TG1 strains - lawn of bacteria on plates</li>
+
           <li>Transformation of chromoprotein BioBrick (Bba_K1033910) using <em>E. coli</em> BL21 and TG1 strains - lawn of bacteria on plates</li>
−
           <li>colony PCR of plated cells with chromoprotein insert - no insert could be multiplied</li>
+
           <li>Colony PCR of plated cells with chromoprotein insert - no insert could be multiplied</li>
−
           <li>transformation of ligated devices ER-ɑ, NahR-GFP1-9, NahR-sfGFP, both plated and also inoculated to 10 mL LB media</li>
+
           <li>Transformation of ligated devices ER-ɑ, NahR-GFP1-9, NahR-sfGFP, both plated and also inoculated to 10 mL LB media</li>
−
           <li>no growth of the three transformed devices on a plate, very low OD as well </li>
+
           <li>No growth of the three transformed devices on a plate, very low OD as well </li>
−
           <li>digestion/ligation of ER-ɑ</li>
+
           <li>Digestion/ligation of ER-ɑ</li>
 
         </ul>
 
         </ul>
 
       </article>
 
       </article>
Line 216: Line 216:
 
         <p class="date">Week 42 (2017/10/16 - 2017/10/22)</p>
 
         <p class="date">Week 42 (2017/10/16 - 2017/10/22)</p>
 
         <ul>
 
         <ul>
−
           <li>transformation of ER-ɑ to <em>E. coli</em> BL21, GFP1-9 and sfGFP (already in a pETDuet-1 plasmid) to both <em>E. coli</em> BL21 and TG1 - no growth (ER-ɑ) or lawn of bacteria (inserts in a pETDuet-1 plasmid)</li>
+
           <li>Transformation of ER-ɑ to <em>E. coli</em> BL21, GFP1-9 and sfGFP (already in a pETDuet-1 plasmid) to both <em>E. coli</em> BL21 and TG1 - no growth (ER-ɑ) or lawn of bacteria (inserts in a pETDuet-1 plasmid)</li>
−
           <li>digestion of GFP1-9 device, ER-ɑ part and ER-ɑ device</li>
+
           <li>Digestion of GFP1-9 device, ER-ɑ part and ER-ɑ device</li>
−
           <li>agarose gel electrophoresis - no bands for GFP1-9</li>
+
           <li>Agarose gel electrophoresis - no bands for GFP1-9</li>
−
           <li>repeated transformation of chromoprotein BioBrick to <em>E. coli</em> DH5-ɑ, BL21 and TG1 strains - no growth</li>
+
           <li>Repeated transformation of chromoprotein BioBrick to <em>E. coli</em> DH5-ɑ, BL21 and TG1 strains - no growth</li>
−
           <li>replating of transformed devices in pETDuet-1 plasmid (ER-ɑ, GFP1-9 and sfGFP) - growth on all plates</li>
+
           <li>Replating of transformed devices in pETDuet-1 plasmid (ER-ɑ, GFP1-9 and sfGFP) - growth on all plates</li>
−
           <li>replated cell colonies were inoculated to 5 mL LB media over night </li>
+
           <li>Replated cell colonies were inoculated to 5 mL LB media over night </li>
−
           <li>digestion of GFP1-9 for agarose gel - smeary bands</li>
+
           <li>Digestion of GFP1-9 for agarose gel - smeary bands</li>
−
           <li>repeated transformation of GFP1-9 device, ER-ɑ part and ER-ɑ device in a pETDuet-1 to <em>E. coli</em> DH5ɑ using a standard and quick transformation protocol</li>
+
           <li>Repeated transformation of GFP1-9 device, ER-ɑ part and ER-ɑ device in a pETDuet-1 to <em>E. coli</em> DH5ɑ using a standard and quick transformation protocol</li>
−
           <li>results: standard protocol - many colonies, quick protocol - lawn of bacteria</li>
+
           <li>Results: standard protocol - many colonies, quick protocol - lawn of bacteria</li>
−
           <li>digestion and ligation of GFP1-9 part and device, ER-ɑ part and device</li>
+
           <li>Digestion and ligation of GFP1-9 part and device, ER-ɑ part and device</li>
−
           <li>inoculation of cells containing GFP1-9 device, sfGFP and B21 competent cells to 5 mL LB media</li>
+
           <li>Inoculation of cells containing GFP1-9 device, sfGFP and B21 competent cells to 5 mL LB media</li>
−
           <li>freezing and thawing of the overnight culture</li>
+
           <li>Freezing and thawing of the overnight culture</li>
 
           <li>SDS-PAGE to check for the expression of the proteins</li>
 
           <li>SDS-PAGE to check for the expression of the proteins</li>
−
           <li>transformation of the samples being ligated overnight using quick and standard protocol</li>
+
        </ul>
−
           <li>colony PCR to check wether ligation succeeded</li>
+
      </article>
−
           <li>agarose gel electrophoresis for checking if the inserts are of correct length</li>
+
      <article class="card">
−
           <li>noticed excessive growth on plates with ampicillin, where there should not be any growth - inoculation of LB media with cells containing different devices, by using both antibiotics, to see where the growth will appear</li>
+
        <p class="date"> Week 43 (2017/10/23 - 2017/10/29)</p>
−
           <li>inoculation of LB media containing ampicillin and chloramphenicol with competent cells to see if the antibiotics were the problem </li>
+
          <ul>
−
           <li>purification of plasmids carrying GFP1-9</li>
+
           <li>Transformation of the samples being ligated overnight using quick and standard protocol</li>
−
           <li>digestio of the latter plasmids with one and two restriction enzymes </li>
+
           <li>Colony PCR to check wether ligation succeeded</li>
−
           <li>agarose gel electrophoresis to check the result of the digestion </li>
+
           <li>Agarose gel electrophoresis for checking if the inserts are of correct length</li>
−
           <li>repeated digestion for much longer time</li>
+
           <li>Noticed excessive growth on plates with ampicillin, where there should not be any growth - inoculation of LB media with cells containing different devices, by using both antibiotics, to see where the growth will appear</li>
−
           <li>colony PCR of purified plasmids</li>
+
           <li>Inoculation of LB media containing ampicillin and chloramphenicol with competent cells to see if the antibiotics were the problem </li>
−
           <li>agarose gel electrophoresis for checking the presence of inserts</li>
+
           <li>Purification of plasmids carrying GFP1-9</li>
 +
           <li>Digestion of the latter plasmids with one and two restriction enzymes </li>
 +
           <li>Agarose gel electrophoresis to check the result of the digestion </li>
 +
           <li>Repeated digestion for much longer time</li>
 +
           <li>Colony PCR of purified plasmids</li>
 +
           <li>Agarose gel electrophoresis for checking the presence of inserts</li>
 
         </ul>
 
         </ul>
 
       </article>
 
       </article>

Latest revision as of 00:52, 2 November 2017

Notebook

June

Week 26 - 2017/06/28 - 2017/06/30

  • Obtaining the pETDuet-1 plasmid backbone
  • Cell cultivation (E. coli BL21-DE3) with pETDuet-1 plasmid
  • Plasmid isolation (pETDuet-1) using GeneJET Plasmid Miniprep Kit
  • Spectrophotometric analysis of plasmids
  • Too low plasmid concentration, therefore we ordered commercial plasmid backbones

July

Week 27 (2017/07/03 - 2017/07/07)

  • Cell cultivation (E. coli BL21 and E. coli TG1)
  • Preparation of competent cells using the Inoue Method for both E. coli strains
  • Freezing of competent cells

Week 28 (2017/07/10 - 2017/07/14)

InterLab attempt 1
  • Transformation of competent (E. coli DH5ɑ) cells
  • The transformation did not succeed
  • Competent cell test (E. coli TG1 and BL21) using Competent Cell Test Kit
  • Successful competent TG1 and BL21 E. coli cells
  • Have not calculated the cell efficiency, as cell competency test was only a test experiment

Week 29 (2017/07/17 - 2017/07/21)

InterLab attempt 2
  • Plating of competent cells (E. coli DH5ɑ)
  • Competent cells grew poorly, therefore non-competent ones were plated as well

Week 30 (2017/07/24 - 2017/07/28)

  • Competent cell test (E. coli DH5ɑ)
  • Preparing competent cells from the non-competent batch of E. coli DH5ɑ cells
  • Low effectiveness of the competent cells was measured
  • The commercial pETDUet-1 plasmids have arrived

August

Week 31 (2017/07/31 - 2017/08/04)

  • The pETDuet-1 plasmids were sent for gene synthesis
  • Transformation of competent E. coli DH5ɑ cells
  • The transformation did not succeed again, we suspect the cells were in a bad condition
  • Asked iGEM team from Gothenburg, to get some E. coli DH5ɑ cells

Week 32 (2017/08/07 - 2017/08/11)

  • The second batch of pETDuet-1 plasmids arrived
  • Received DH5ɑ cells from iGEM team Chalmers Gothenburg
  • Making DH5ɑ competent cells

Week 33 (2017/08/14 - 2017/08/18)

  • pETDuet-1 plasmids sent to Eurofins
  • Competent cell test (E. coli DH5ɑ, BL21, NEB5ɑ)
  • Transformation of competent E. coli BL21 cells with test BioBricks
  • The transformed cells for the InterLab study did not grow
  • Contacted iGEM Headquarters to send us more plasmid DNA
  • Contacted iGEM DTU Denmark to send us already transformed DH5ɑ cells for the InterLab study

Week 34 (2017/08/21 - 2017/08/25)

  • Plasmid isolation from transformed E. coli BL21 cells with test BioBricks
  • Restriction digestion
  • Agarose gel electrophoresis

September

Week 35 (2017/08/28 - 2017/09/03)

  • Gel extraction of test BioBricks
  • Digestion of the plasmid backbone and ligation of devices 1G1 and 1G2
  • Agarose gel electrophoresis
  • Transformation, which resulted in red (containing RFP) and white cells (should not be there)
  • Inoculation  of the media with both red and white cells growing after transformation to get more DNA to send for sequencing
  • Plasmid isolation and freezing of DNA
  • Repeating InterLab with cells from iGEM DTU Denmark
  • InterLab measurements resulted in negative values
  • Repeated the plating of the cells by concentrating already grown bacterial culture
  • After 26 h incubation no cells grew  

Week 37 (2017/09/11 - 2017/09/17)

  • Digestion and ligation of gBlocks GFP1-9 and gBlock GFP1-9 device
  • Agarose gel electrophoresis to see how successful the ligation was
  • Transformation of the ligated gBlocks to E. coli BL21
  • Transformation of E. coli DH5ɑ cells with the new batch of devices
  • Plating of the transformed cells
  • Preparing 10 mL culture media

Week 38 (2017/09/18 - 2017/09/25)

  • Inoculation of 100 mL media with the overnight culture
  • InterLab measurements
  • Digestion and ligation of the GFP1-9 device and GFP1-9; ligation was done overnight
  • Digestion and ligation of devices for interlab; ligation was done overnight
  • Colony PCR of InterLab devices to check if the colonies appearing white on the media had GFP inserted
  • PCR products were run on agarose gel

October

Week 39 (2017/09/25 - 2017/10/01)

  • InterLab measurements
  • Preparing glycerol stocks of E. coli BL21 cells with inserted GFP1-9 and GFP1-9 device
  • Plating of cells from the glycerol stocks to check if they were prepared correctly
  • Digestion and ligation of the NahR BioBrick
  • Transformation of NahR BioBrick to E. coli BL21
  • Colony PCR of the transformed NahR BioBrick
  • Plasmid purification of GFP1-9 and GFP1-9 device BioBricks

Week 40 (2017/10/02 - 2017/10/08)

  • Harvesting the cells cultivated from the glycerol stock
  • Sonication of cells with the GFP1-9 device insert
  • Plating cells with GFP1-9 device insert to measure any possible background fluorescence
  • Running SDS-PAGE to check for expression of GFP1-9
  • Digestion and ligation of NahR-GFP1-9 device and NahR-sfGFP device
  • Transformation of ligated NahR-GFP1-9 device and NahR-sfGFP device
  • Transformation of NahR in pETDuet vector to E. coli BL21, plating of the cells - no growth
  • Agarose gel electrophoresis to check if the ligation succeeded - it did not
  • Repeated digestion and ligation of NahR-GFP1-9 device and NahR-sfGFP device
  • Repeated transformation of ligated NahR-GFP1-9 device and NahR-sfGFP device

Week 41 (2017/10/09 - 2017/10/15)

  • Colony PCR of the plated GFP1-9 device
  • Plating of competent E. coli BL21 cells and E. coli BL21 cells with GFP1-9 insert from the glycerol stock
  • Digestion and ligation of ER-ɑ device, NahR-GFP1-9  device and NahR-sfGFP device
  • Agarose gel electrophoresis of digested ER-ɑ device, NahR-GFP1-9  device and NahR-sfGFP device products
  • Transformation of chromoprotein BioBrick (Bba_K1033910) using E. coli BL21 and TG1 strains - lawn of bacteria on plates
  • Colony PCR of plated cells with chromoprotein insert - no insert could be multiplied
  • Transformation of ligated devices ER-ɑ, NahR-GFP1-9, NahR-sfGFP, both plated and also inoculated to 10 mL LB media
  • No growth of the three transformed devices on a plate, very low OD as well
  • Digestion/ligation of ER-ɑ

Week 42 (2017/10/16 - 2017/10/22)

  • Transformation of ER-ɑ to E. coli BL21, GFP1-9 and sfGFP (already in a pETDuet-1 plasmid) to both E. coli BL21 and TG1 - no growth (ER-ɑ) or lawn of bacteria (inserts in a pETDuet-1 plasmid)
  • Digestion of GFP1-9 device, ER-ɑ part and ER-ɑ device
  • Agarose gel electrophoresis - no bands for GFP1-9
  • Repeated transformation of chromoprotein BioBrick to E. coli DH5-ɑ, BL21 and TG1 strains - no growth
  • Replating of transformed devices in pETDuet-1 plasmid (ER-ɑ, GFP1-9 and sfGFP) - growth on all plates
  • Replated cell colonies were inoculated to 5 mL LB media over night
  • Digestion of GFP1-9 for agarose gel - smeary bands
  • Repeated transformation of GFP1-9 device, ER-ɑ part and ER-ɑ device in a pETDuet-1 to E. coli DH5ɑ using a standard and quick transformation protocol
  • Results: standard protocol - many colonies, quick protocol - lawn of bacteria
  • Digestion and ligation of GFP1-9 part and device, ER-ɑ part and device
  • Inoculation of cells containing GFP1-9 device, sfGFP and B21 competent cells to 5 mL LB media
  • Freezing and thawing of the overnight culture
  • SDS-PAGE to check for the expression of the proteins

Week 43 (2017/10/23 - 2017/10/29)

  • Transformation of the samples being ligated overnight using quick and standard protocol
  • Colony PCR to check wether ligation succeeded
  • Agarose gel electrophoresis for checking if the inserts are of correct length
  • Noticed excessive growth on plates with ampicillin, where there should not be any growth - inoculation of LB media with cells containing different devices, by using both antibiotics, to see where the growth will appear
  • Inoculation of LB media containing ampicillin and chloramphenicol with competent cells to see if the antibiotics were the problem
  • Purification of plasmids carrying GFP1-9
  • Digestion of the latter plasmids with one and two restriction enzymes
  • Agarose gel electrophoresis to check the result of the digestion
  • Repeated digestion for much longer time
  • Colony PCR of purified plasmids
  • Agarose gel electrophoresis for checking the presence of inserts