Team:NTNU Trondheim/Notebook

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<h3 class="centered">Week 24</h3>
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<h3 class="centered">Week 25</h3>
<h5 class="centered">(16/06 - 22/06)</h5>
<h5 class="centered">(16/06 - 22/06)</h5>

Revision as of 11:48, 12 August 2014

Team:NTNU_Trondheim/notebook - 2014.igem.org

 

Team:Cornell/notebook

From 2013.igem.org

NTNU Genetically Engineered Machines

Notebook

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Week 23

(02/06 - 08/06)

June 3rd
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  1. Prepared SOC and yB solutions for future lab work.
  2. Sterilized material and solutions needed for future lab work.

June 4th
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{{{tech}}}

Made LB plates with ampicillin and ampicillin + kanamycin.

June 5th
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{{{tech}}}

Inoculated E. coli DH5α in SOC medium overnight.

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

Made competent E. coli DH5α cells.

Week 24

(09/06 - 15/06)

June 10th
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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
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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.

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
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The SOC medium was clear the next day, meaning no growth.

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

June 13th
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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
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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
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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
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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.

  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
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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
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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.