Team:NTNU Trondheim/Notebook

From 2014.igem.org

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                         <li>Prepared <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#2">SOC</a> and <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#3">yB</a> solutions for future lab work.</li>
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                         <li>Prepared SOC and yB solutions for future lab work.</li>
                         <li>Sterilized material and solutions needed for future lab work.</li>
                         <li>Sterilized material and solutions needed for future lab work.</li>
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<p> Made <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#1">LB plates</a> with ampicillin and ampicillin + kanamycin.
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<p> Made LB plates with ampicillin and ampicillin + kanamycin.
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<p> Inoculated <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#21"><i>E. coli</i> DH5α</a> in <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#2">SOC</a> medium overnight.
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<p> Inoculated <i>E. coli DH5α</i> in SOC medium overnight.
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<div class="nb-tech">OD of culture was 0.3160 after 100 minutes.</div>
<div class="nb-tech">OD of culture was 0.3160 after 100 minutes.</div>
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<p> Made <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-molecular/heat-shock-transformation-2">competent <i>E. coli</i></a> DH5α cells.
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<p> Made competent <i>E. coli</i> DH5α cells.
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<div class="nb-onetech-i nohilite">show technical details</div>
<div class="nb-onetech-i nohilite">show technical details</div>
</h6>
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<div class="nb-tech">A plasmid containing ampicillin resistance was used for the transformation, and the cells were incubated overnight on <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#1">LB plates</a> with ampicillin. Plates showed a bacterial blanket the next day; the cells were apparently super competent.</div>
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<div class="nb-tech">Transformation was done by heat-shock. A plasmid containing ampicillin resistance was used, and the transformed cells were incubated overnight on LB plates with ampicillin. Plates showed a bacterial blanket the next day; the cells were apparently super competent.</div>
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<p> Test of <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#13">heat-shock transformation</a> <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#23">efficiency</a> of competent <i>E. coli</i> DH5α from June 6th.  
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<p> Test of transformation efficiency of competent <i>E. coli</i> DH5α from June 6th.  
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<div class="nb-onetech-i nohilite">show technical details</div>
</h6>
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<div class="nb-tech">Only 1 µl was used for the transformation, the rest of the BioBrick stock solution was stored -20 °C. <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> was located at plate 4, 17F. <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> was located at plate 4, 1N and <a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a> was located at plate 4, 1P. The J BioBrick is a promotor region, B BioBrick is an RBS region and C BioBrick is LacI repressor gene. Plates showed decent growth the next day; transformation was a success.</div>
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<div class="nb-tech">Only 1 µl was used for the transformation, the rest of the BioBrick stock solution was stored -20 °C. BBa_J23101 was located at plate 4, 17F. BBa_B0034 was located at plate 4, 1N and BBa_C0012 was located at plate 4, 1P. Plates showed decent growth the next day; transformation a success. The J BioBrick was meant to be used as a promotor for B and C BioBrick. B BioBrick is an RBS region and C BioBrick is the LacI repressor gene.</div>
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<p> Rehydrated BioBrick <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a>, and <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#13">heat-shock transformed</a> them into competent <i>E. coli</i> DH5α cells, and incubated the cultures on <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#1">LB plates</a> with ampicillin overnight.
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<p> Rehydrated BioBrick BBa_J23101, BBa_B0034 and BBa_C0012, and transformed them into competent <i>E. coli</i> DH5α cells by heat-shock, and incubated the cultures on LB plates with ampicillin overnight.
</p>
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<div class="nb-onetech-i nohilite">show technical details</div>
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<div class="nb-tech">The medium was clear the next day, indicating no growth. </div>
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<div class="nb-tech">The SOC medium was clear the next day, meaning no growth. </div>
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<p> Inoculated BioBrick colonies from June 11th in <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#2">SOC</a> medium containing ampicillin.  
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<p> Inoculated BioBrick colonies from June 11th in SOC medium containing ampicillin.  
</p>
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<div class="nb-tech">The failed inoculation attempt on June 12th could indicate something wrong with the LB plates with ampicillin. Growth of non-transformed cells supported this. Most likely the ampicillin was aliquotted to the medium at a temperature causing the antibiotic to denature. </div>
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<div class="nb-tech">The failed inoculation attempt on June 12th could indicate something wrong with the LB plates with ampicillin. Growth of non-transformed cells supported this. Most likely the ampicillin was aliquotted to the medium at an elevated temperature, causing denaturation of the antibiotic. </div>
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<p> Negative control of non-transformed <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#21"><i>E. coli</i> DH5α</a> on <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#1">LB plates</a> with ampicillin.  
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<p> Negative control of non-transformed <i>E. coli</i> DH5α on LB plates with ampicillin.  
</p>
</p>
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<div class="nb-tech">The negative control showed no growth, meaning the LB plates with ampicillin + kanamycin were made correctly! Plates with J, B and C BioBricks showed reasonable growth the next day.</div>
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<div class="nb-tech">The negative control showed no growth, meaning the LB plates with ampicillin + kanamycin were properly made. Plates with J, B and C BioBricks showed reasonable growth the next day.</div>
<p> <ol>  
<p> <ol>  
-
<li>Negative control of non-transformed <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#21"><i>E. coli</i> DH5α</a> on <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#1">LB plates</a> with ampicillin + kanamycin.</li>
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                        <li>Negative control of non-transformed <i>E. coli</i> DH5α on LB plates with ampicillin + kanamycin.</li>
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            <li>Made new LB plates with ampicillin.</li>
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                        <li>Made new LB plates with ampicillin.</li>
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<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#13">Heat-shock transformed</a> <i>E. coli</i> DH5α with BioBricks <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a> and plated onto LB with ampicillin.</li>
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<li>Transformed <i>E. coli</i> DH5α with BioBricks BBa_J23101, BBa_B0034 and BBa_C0012 and plated onto LB with ampicillin.</li>
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</ol>
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</p>
</p>
</div>
</div>
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<div class="nb-onetech-i nohilite">show technical details</div>
<div class="nb-onetech-i nohilite">show technical details</div>
</h6>
</h6>
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<div class="nb-tech">Left_flank and Right_flank sequences were needed to achieve homologous recombination of insert into <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#22"><i>Synechocystis</i> PCC. δslr0906</a> genome. Kanamycin_resistance optimized for <i>Synechocystis</i> was needed as screening mechanism in order to select for transformed colonies. Successful PCR amplification of Left_flank (556 bp) and Kanamycin_resistance (944 bp) sequence. Right_flank (544 bp) sequence amplification failed.</div>
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<div class="nb-tech">These DNA sequences were to be used in the final construct. Left_flank and Right_flank sequences were needed to make conjugation of construct with the genome of <i>Synechocystis</i> PCC. 6803 possible. Kanamycin_resistance optimized for <i>Synechocystis</i> was needed in order to select for colonies with conjugated final construct. Successful PCR amplification of Left_flank (556 bp) and Kanamycin_resistance (944 bp) sequence. Right_flank (544 bp) sequence amplification failed.</div>
<p> <ol>
<p> <ol>
-
<li>PCR amplification of <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#17">Left_flank</a>, <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#16">Righ_flank</a> and <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#18">Kanamycin_resistance</a> with <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#22"><i>Synechocystis</i> PCC. δslr0906</a> as template using touchdown <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#9">PCR</a>. </li>
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<li>PCR amplification of Left_flank, Righ_flank and Kanamycin_resistance with <i>Synechocystis</i> PCC. δslr0906 as template using touchdown PCR. </li>
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<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#15">Gel electrophoresis verification</a> of PCR products on agarose gel</li>
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<li>Verification of PCR products on agarose gel</li>
</ol>
</ol>
</p>
</p>
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<div class="nb-onetech-i nohilite">show technical details</div>
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</h6>
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<div class="nb-tech">J (35 bp), B (12 bp) and C (1153 bp) BioBricks all showed proper band lengths after verification on agarose gel. Note that it is not possible to physically see B and J on the gel, but the backbone attached to B and J could be seen. The mCherry gene did not show as a band on the gel, possibly due to incompatibilities between the mCherry gene and the primers. The mCherry gene was to be used as a marker gene, giving visual confirmation of successful transformation; however, because of the possible compatibility issues we decided to explore other options. In the end we decided to use RFP as marker.</div>
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<div class="nb-tech">J (35 bp), B (12 bp) and C (1153 bp) BioBricks all showed proper band lengths after verification on agarose gel. Note that it is not possible to physically see B and J on the gel, but the backbone attached to B and J is shown. The mCherry gene did not show any band on the gel, suggesting incompatibilities between the mCherry gene and the primers. Originally the mCherry gene was meant to be used as a visual confirmation of successful transformation of the final construct, but because compatibility issues with the primers we decided to explore other options. In the end we decided to use a red fluorescent protein as marker.</div>
<p> <ol>
<p> <ol>
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<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#7">Mini-prep</a> of PCR products from June 17th.</li>
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<li>Mini-prep of PCR products from June 17th.</li>
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<li>PCR amplification of <a href="http://www.snapgene.com/resources/plasmid_files/fluorescent_protein_genes_and_plasmids/mCherry/">mCherry</a> gene, BioBricks <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a> using touchdown <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#9">PCR</a>.</li>
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<li>PCR amplification of mCherry gene, BioBricks BBa_J23101, BBa_B0034 and BBa_C0012 using touchdown PCR.</li>
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<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#15">Gel electrophoresis verification</a> of PCR products on agarose gel</li>
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<li>Verification of PCR products on agarose gel</li>
</ol>
</ol>
</p>
</p>
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<div class="nb-tech">J and B BioBricks were digested with <i>Xba</i>I, while C BioBrick was digested with <i>Not</i>I-HF. Digest of J, B and C should have gievn a band of 2989 bp, 2097 bp and 2063 bp respectively on the gel; low concentration of DNA after digest, but all samples had correct band lengths.</div>
<div class="nb-tech">J and B BioBricks were digested with <i>Xba</i>I, while C BioBrick was digested with <i>Not</i>I-HF. Digest of J, B and C should have gievn a band of 2989 bp, 2097 bp and 2063 bp respectively on the gel; low concentration of DNA after digest, but all samples had correct band lengths.</div>
<p> <ol>
<p> <ol>
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<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#8">Digest</a> of BioBricks <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a>.</li>
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<li>Digest of BioBricks BBa_J23101, BBa_B0034 and BBa_C0012.</li>
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<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#15">Gel electrophoresis verification</a> of digest on agarose gel.</li>
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<li>Verification of digest on agarose gel.</li>
</ol>
</ol>
</p>
</p>
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<div class="nb-tech">The upstream part (J) was cut with <i>Eco</i>RI-HF and <i>Spe</i>I restriction enzymes, the downstream part (B) was cut with <i>Xba</i>I and <i>Pst</i>I-HF restriction enzymes, while the psB1C3 backbone was cut with <i>Eco</i>RI-HF, <i>Pst</i>I-HF and <i>Dpn</i>I restriction enzymes. The restriction mixtures were left at 37 °C for one hour, heat killed at 80 °C for 30 minutes, then stored at -20 °C.</div>
<div class="nb-tech">The upstream part (J) was cut with <i>Eco</i>RI-HF and <i>Spe</i>I restriction enzymes, the downstream part (B) was cut with <i>Xba</i>I and <i>Pst</i>I-HF restriction enzymes, while the psB1C3 backbone was cut with <i>Eco</i>RI-HF, <i>Pst</i>I-HF and <i>Dpn</i>I restriction enzymes. The restriction mixtures were left at 37 °C for one hour, heat killed at 80 °C for 30 minutes, then stored at -20 °C.</div>
<p> <ol>
<p> <ol>
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<li>Made <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#1">LB plates</a> with chloramphenicol.</li>
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<li>Made LB plates with chloramphenicol.</li>
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<li>Digest of <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:pSB1C3">psB1C3 backbone</a> according to the <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#11">3A assembly</a> method.</li>
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<li>Digest of BBa_J23101, BBa_B0034 and psB1C3 plasmid backbone according to the 3A Assemble method.</li>
</ol>
</ol>
</p>
</p>
</div>
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<h3 class="centered">Week 26</h3>
 
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<h5 class="centered">(23/06 - 29/06)</h5>
 
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</p>
 
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<div class="entry nb-wet">
 
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<h6>June 23rd
 
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<div class="nb-tech">Ligation was performed at 42 °C for 20 minutes using <i>Taq</i> ligase with ratio 3 between amount of insert and backbone. The ligation mixture was split into two aliquots (1 µl and 9 µl) and transformed into competent <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#21"><i>E. coli</i> DH5α</a> at 45 °C and plated out on <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#1">LB plates</a> with chloramphenicol. The plates showed no growth the next day. Negative control of the chloramphenicol plates did not show growth either, meaning that the plates were not the issue. Other reasons might be: (1) using <i>Taq</i> ligase instead of <i>T4</i> ligase might alter the ligation efficiency, and hence the transformation efficiency; however, somw growth would still be expected; (2) issues with the enzymatic digestion. According to the <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#11">3A assembly</a> method, the downstream part and the backbone is supposed to be cut with PstI, not PstI-HF. PstI-HF is heat resistant and cannot be heat killed. The remaining enzymatic activity of PstI-HF could therefore have disrupted the <i>Taq</i> ligase activity. However, freezing should have inactivated PstI-HF activity, meaning it is not likely to cause problems; and (3) low concentrations of B and J. NanoDrop measured B at 5.3 ng/µl concentration and J at 10.1 ng/µl concentration.</div>
 
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<p> <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#12">Ligation</a> and <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#13">heat-shock transformation</a> of digestion mixtures from June 20th.
 
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<h6>June 24th
 
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</h6>
 
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<div class="nb-tech">Variations from June 23rd included: (1) the ligation was performed at 45 °C for 20 minutes and (2) the entire ligation mixture (10 µl) was transformed into one aliquot of competent <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#21"><i>E. coli</i> DH5α</a> cells; however, ligation still failed.
 
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<br>The gel verification of digest showed an unexpected band of the J BioBrick, and based on failed ligation attempts, it was decided to inoculate new BioBrick colonies.</div>
 
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<p> <ol>
 
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<li>Attempted <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#12">ligation</a> and <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#13">heat-shock transformation</a> of the digestion mixtures from June 20th again.</li>
 
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<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#15">Gel electrophoresis verification</a> of digest from June 20th on agarose gel.</li>
 
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<li>Inoculated new colonies of <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a>.
 
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<h6>June 25th
 
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</h6>
 
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<div class="nb-tech">Enzymatic digestion performed with similar procedure as June 20th: upstream part (J) cut with <i>Eco</i>RI-HF and <i>Spe</i>I, downstream part (B) cut with <i>Xba</i>I and <i>Pst</i>I-HF and psB1C3 backbone cut with <i>Eco</i>RI-HF and <i>Pst</i>I-HF. Digestion mixtures were left at 37 °C for one hour, then heat-killed at 80 °C for 30 minutes, before being stored at -20 °C.
 
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<br>Verification of Left_flank, Right_flank and Kanamycin_resistance on gel showed expected bands at 566 bp, 552 bp and 961 bp respectively. </div>
 
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<p> <ol>
 
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<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#7">Mini-prep</a> of BioBrick colonies from June 24th.</li>
 
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<li>Enzymatic digestion of newly mini-prepped <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:pSB1C3">psB1C3 backbone</a>.</li>
 
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<li>New PCR amplification of <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#17">Left_flank</a>, <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#16">Righ_flank</a> and <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#18">Kanamycin_resistance</a> using touchdown <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#9">PCR</a>.</li>
 
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<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#15">Gel electrophoresis verification</a> of PCR products on agarose gel.</li>
 
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</ol>
 
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</p>
 
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<h6>June 26th
 
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<div class="nb-onetech-i nohilite">show technical details</div>
 
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</h6>
 
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<div class="nb-tech">The problem with gel verification of B and J is the length of B and J which is 12 bp and 35 bp respectively. They are too short to appear on the gel, and it is therefore impossible to whether or not the samples contain the correct sequence of DNA. However, because these are BioBricks, it is assumed the transformed <i>E. coli</i> DH5α cells contain correct plasmids.
 
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<br>The ligation procedure was performed as June 23rd with a few exceptions: (1) increased the volume of ligation mixture, and hence the amount of ligase. This was because it was assumed that the residual activity of <i>Pst</i>I-HF could be counteracted by increasing <i>Taq</i> ligase concentration; and (2) created a short time series for ligation, one sample at 42 °C for 20 minutes and another one for one hour. Both ligation times resulted in growth on <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#1">LB plates</a> with chloramphenicol.</div>
 
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<p> <ol>
 
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<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#15">Gel electrophoresis verification</a> of <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:pSB1C3">psB1C3 backbone</a> backbone digest.</li>
 
-
<li>Another attempted <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#12">ligation</a> of digested B, J and psB1C3 backbone from June 25th, and <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#13">heat-shock transformation</a> into competent <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#21"><i>E. coli</i> DH5α</a> cells.</li>
 
-
</ol>
 
-
</p>
 
-
</div>
 
-
</div>
 
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<div class="entry nb-wet">
 
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<div>
 
-
<h6>June 27th
 
-
<div class="nb-onetech-i nohilite">show technical details</div>
 
-
</h6>
 
-
<div class="nb-tech">The ligation procedure was performed as June 26th, except that the ligation time was 20 minutes. Transformation into competent <i>E. coli</i> DH5α cells followed standard protocol. The next day two colonies were observed on the plate.</div>
 
-
<p> <ol>
 
-
<li>QIAquick PCR purification of <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#17">Left_flank</a>, <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#16">Righ_flank</a> and <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#18">Kanamycin_resistance</a> PCR product from June 25th.</li>
 
-
<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#8">Digested</a> and <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#12">ligated</a> Right_Flank, Kanamycin_resistance and <a href="http://parts.igem.org/Part:pSB1A3">psB1A3 backbone</a>, then <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#13">heat-shock transformed</a> it into competent <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#21"><i>E. coli</i> DH5α</a> cells.</li>
 
-
<li>Inoculated colonies from ligation of <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:pSB1C3">psB1C3 backbone</a> from June 26th.</li>
 
-
</ol>
 
-
</p>
 
-
</div>
 
-
</div>
 
-
 
-
<div class="entry nb-wet">
 
-
<div>
 
-
<h6>June 28th
 
-
<div class="nb-onetech-i nohilite">show technical details</div>
 
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</h6>
 
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<div class="nb-tech">{{{tech}}}</div>
 
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<p> <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#7">Mini-prep</a> of inoculated colonies from <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:pSB1C3">psB1C3 backbone</a> ligation on June 26th.
 
-
</p>
 
-
</div>
 
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</div>
 
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<p>
 
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-
<div id="week27entry" class="nb-week">
 
-
<div class="twelve columns">
 
-
<h3 class="centered">Week 27</h3>
 
-
<h5 class="centered">(30/06 - 06/07)</h5>
 
-
 
-
</p>
 
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-
<div class="entry nb-wet">
 
-
<div>
 
-
<h6>June 30th
 
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<div class="nb-onetech-i nohilite">show technical details</div>
 
-
</h6>
 
-
<div class="nb-tech">. The gel verification of the ligation on June 26th showed only one band corresponding to the length of a backbone (~2000 bp). Normally, the gel would run for 50 minutes at 80 V, but because of the length of JB (47 bp), it was set to run only 30 minutes. However, despite the shorter run-time, no band corresponding to JB was seen. GelGreen binds DNA and visualises it. We assume that shorter DNA segments binds less GelGreen, which would make the absence of a JB band on the gel understandable. We therefore assumed that the JB ligation was a success despite the failed verification. JB ligation to LF and psB1A3 backbone showed growth the next day.</div>
 
-
<p> <ol>
 
-
<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#15">Gel electrophoresis verification</a> of <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:pSB1C3">psB1C3 backbone</a> ligation on agarose gel.</li>
 
-
<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#8">Digestion</a>, <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#12">ligation</a> and <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#13">heat-shock transformation</a> of the ligated JB with <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#17">Left_flank</a> and <a href="http://parts.igem.org/Part:pSB1A3">psB1A3 backbone</a>.</li>
 
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</ol>
 
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</p>
 
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</div>
 
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</div>
 
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<div class="entry nb-wet">
 
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<div>
 
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<h6>July 1st
 
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<div class="nb-onetech-i nohilite">show technical details</div>
 
-
</h6>
 
-
<div class="nb-tech">A different enzyme was used for the gel verification process because gel verification thus far had been prone to errors. We speculated that the difference in enzyme activity of <i>Eco</i>RI-HF, <i>Pst</i>I-HF, <i>Spe</i>I and <i>Xba</i>I could have caused uneven cutting of the plasmids, which could have produced erroneous bands.
 
-
<br>The agarose gel of <i>Not</i>I-HF digested {RF, Kan, psB1A3} and {J,B, psB1A3} showed bands at around 1000 bp for all samples, which is wrong in both cases.</div>
 
-
<p> <ol>
 
-
<li>New <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#15">gel electrophoresis verification</a> of <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:pSB1C3">psB1C3 backbone</a> ligation on agarose gel using <i>Not</i>I-HF restriction enzyme.</li>
 
-
<li>Also verified <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#16">Righ_flank</a>, <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#18">Kanamycin_resistance</a> and <a href="http://parts.igem.org/Part:pSB1A3">psB1A3 backbone</a> ligation from June 27th on agarose gel using <i>Not</i>I-HF.</li>
 
-
<li>Inoculation of <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#17">Left_flank</a>, JB and psB1A3 backbone colonies from June 30th.</li>
 
-
</ol>
 
-
</p>
 
-
</div>
 
-
</div>
 
-
 
-
<div class="entry nb-wet">
 
-
<div>
 
-
<h6>July 2nd
 
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<div class="nb-onetech-i nohilite">show technical details</div>
 
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</h6>
 
-
<div class="nb-tech">All samples showed erroneous bands on the agarose gel, and it was therefore decided to restart the whole ligation process.</div>
 
-
<p> <ol>
 
-
<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#7">Mini-prep</a> of {LF, JB, psB1A3} from June 30th.</li>
 
-
<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#15">Gel electrophoresis verification</a> of {RF, Kan, psB1A3}, {J,B, psB1A3} and {Left_flank, JB, psB1A3} on agarose gel using <i>Not</i>I-HF restriction enzyme.</li>
 
-
<li>Inoculated new colonies from plates containing colonies from the original <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> and <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> BioBricks.</li>
 
-
</ol>
 
-
</p>
 
-
</div>
 
-
</div>
 
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-
<div class="entry nb-wet">
 
-
<div>
 
-
<h6>July 3rd
 
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<div class="nb-onetech-i nohilite">show technical details</div>
 
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</h6>
 
-
<div class="nb-tech">The gel showed correct band lengths for all samples. J, LF and Kan were digested using <i>Eco</i>RI-HF and <i>Spe</i>I. B and RF were digested using <i>Xba</i>I and <i>Pst</i>I, while the backbones were digested using <i>Eco</i>RI-HF and <i>Pst</i>I. Since it was not possible to heat-kill the high fidelity <i>Pst</i>I enzyme, it was decided to attempt digestion with the non-high fidelity one. This should remove residual enzymatic activity of all restriction enzymes after heat-kill.</div>
 
-
<p> <ol>
 
-
<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#7">Mini-prep</a> of inoculated <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> and <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> colonies from July 2nd.</li>
 
-
<li><a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#15">Gel electrophoresis verification</a> of J and B on an agarose gel using <i>Not</i>I-HF restriction enzyme.</li>
 
-
<li>Digested J, B, <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#17">Left_flank</a>, <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#16">Righ_flank</a>, <a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols#18">Kanamycin_resistance</a>, <a href="http://parts.igem.org/Part:pSB1A3">psB1A3 backbone</a> and <a href="http://parts.igem.org/Part:pSB1C3">psB1C3 backbone</a>.</li>
 
-
</ol>
 
-
</p>
 
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</div>
 
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</div>
 
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<p>
<p>

Revision as of 05:34, 14 October 2014

Team:NTNU_Trondheim/notebook - 2014.igem.org

 

Team:Cornell/notebook

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NTNU Genetically Engineered Machines

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Week 23

(02/06 - 08/06)

June 3rd
show technical details

  1. Prepared SOC and yB solutions for future lab work.
  2. Sterilized material and solutions needed for future lab work.

June 4th
show technical details
{{{tech}}}

Made LB plates with ampicillin and ampicillin + kanamycin.

June 5th
show technical details
{{{tech}}}

Inoculated E. coli DH5α in SOC medium overnight.

June 6th
show technical details
OD of culture was 0.3160 after 100 minutes.

Made competent E. coli DH5α cells.

Week 24

(09/06 - 15/06)

June 10th
show technical details
Transformation was done by heat-shock. A plasmid containing ampicillin resistance was used, and the transformed cells were incubated overnight on LB plates with ampicillin. Plates showed a bacterial blanket the next day; the cells were apparently super competent.

Test of transformation efficiency of competent E. coli DH5α from June 6th.

June 11th
show technical details
Only 1 µl was used for the transformation, the rest of the BioBrick stock solution was stored -20 °C. BBa_J23101 was located at plate 4, 17F. BBa_B0034 was located at plate 4, 1N and BBa_C0012 was located at plate 4, 1P. Plates showed decent growth the next day; transformation a success. The J BioBrick was meant to be used as a promotor for B and C BioBrick. B BioBrick is an RBS region and C BioBrick is the LacI repressor gene.

Rehydrated BioBrick BBa_J23101, BBa_B0034 and BBa_C0012, and transformed them into competent E. coli DH5α cells by heat-shock, and incubated the cultures on LB plates with ampicillin overnight.

June 12th
show technical details
The SOC medium was clear the next day, meaning no growth.

Inoculated BioBrick colonies from June 11th in SOC medium containing ampicillin.

June 13th
show technical details
The failed inoculation attempt on June 12th could indicate something wrong with the LB plates with ampicillin. Growth of non-transformed cells supported this. Most likely the ampicillin was aliquotted to the medium at an elevated temperature, causing denaturation of the antibiotic.

Negative control of non-transformed E. coli DH5α on LB plates with ampicillin.

Week 25

(16/06 - 22/06)

June 16th
show technical details
The negative control showed no growth, meaning the LB plates with ampicillin + kanamycin were properly made. Plates with J, B and C BioBricks showed reasonable growth the next day.

  1. Negative control of non-transformed E. coli DH5α on LB plates with ampicillin + kanamycin.
  2. Made new LB plates with ampicillin.
  3. Transformed E. coli DH5α with BioBricks BBa_J23101, BBa_B0034 and BBa_C0012 and plated onto LB with ampicillin.

June 17th
show technical details
These DNA sequences were to be used in the final construct. Left_flank and Right_flank sequences were needed to make conjugation of construct with the genome of Synechocystis PCC. 6803 possible. Kanamycin_resistance optimized for Synechocystis was needed in order to select for colonies with conjugated final construct. Successful PCR amplification of Left_flank (556 bp) and Kanamycin_resistance (944 bp) sequence. Right_flank (544 bp) sequence amplification failed.

  1. PCR amplification of Left_flank, Righ_flank and Kanamycin_resistance with Synechocystis PCC. δslr0906 as template using touchdown PCR.
  2. Verification of PCR products on agarose gel

June 18th
show technical details
J (35 bp), B (12 bp) and C (1153 bp) BioBricks all showed proper band lengths after verification on agarose gel. Note that it is not possible to physically see B and J on the gel, but the backbone attached to B and J is shown. The mCherry gene did not show any band on the gel, suggesting incompatibilities between the mCherry gene and the primers. Originally the mCherry gene was meant to be used as a visual confirmation of successful transformation of the final construct, but because compatibility issues with the primers we decided to explore other options. In the end we decided to use a red fluorescent protein as marker.

  1. Mini-prep of PCR products from June 17th.
  2. PCR amplification of mCherry gene, BioBricks BBa_J23101, BBa_B0034 and BBa_C0012 using touchdown PCR.
  3. Verification of PCR products on agarose gel

June 19th
show technical details
J and B BioBricks were digested with XbaI, while C BioBrick was digested with NotI-HF. Digest of J, B and C should have gievn a band of 2989 bp, 2097 bp and 2063 bp respectively on the gel; low concentration of DNA after digest, but all samples had correct band lengths.

  1. Digest of BioBricks BBa_J23101, BBa_B0034 and BBa_C0012.
  2. Verification of digest on agarose gel.

June 20th
show technical details
The upstream part (J) was cut with EcoRI-HF and SpeI restriction enzymes, the downstream part (B) was cut with XbaI and PstI-HF restriction enzymes, while the psB1C3 backbone was cut with EcoRI-HF, PstI-HF and DpnI restriction enzymes. The restriction mixtures were left at 37 °C for one hour, heat killed at 80 °C for 30 minutes, then stored at -20 °C.

  1. Made LB plates with chloramphenicol.
  2. Digest of BBa_J23101, BBa_B0034 and psB1C3 plasmid backbone according to the 3A Assemble method.

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