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Team:NTNU Trondheim/Notebook
From 2014.igem.org
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Team:Cornell/notebook
From 2013.igem.org
Notebook
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Week 23
(02/06 - 08/06)
June 3rd
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- Prepared SOC and yB solutions for future lab work.
- Sterilized material and solutions needed for future lab work.
June 4th
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Made LB plates with ampicillin and ampicillin + kanamycin.
June 5th
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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. 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
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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.
- Negative control of non-transformed E. coli DH5α on LB plates with ampicillin + kanamycin.
- Made new LB plates with ampicillin.
- 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.
- PCR amplification of Left_flank, Righ_flank and Kanamycin_resistance with Synechocystis PCC. δslr0906 as template using touchdown PCR.
- 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. 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.
- Mini-prep of PCR products from June 17th.
- PCR amplification of mCherry gene, BioBricks BBa_J23101, BBa_B0034 and BBa_C0012 using touchdown PCR.
- 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.
- Digest of BioBricks BBa_J23101, BBa_B0034 and BBa_C0012.
- 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.
- Made LB plates with chloramphenicol.
- Digest of BBa_J23101, BBa_B0034 and psB1C3 plasmid backbone according to the 3A Assemble method.
