Team:NTNU Trondheim/Notebook

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NTNU Genetically Engineered Machines

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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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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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A plasmid containing ampicillin resistance was used for the transformation, and the 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 heat-shock 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. 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.

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

June 12th
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The medium was clear the next day, indicating 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 a temperature causing the antibiotic to denature.

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 made correctly! 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. Heat-shock 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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Left_flank and Right_flank sequences were needed to achieve homologous recombination of insert into Synechocystis sp. PCC 6803 genome. Kanamycin_resistance optimized for Synechocystis 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.

  1. PCR amplification of Left_flank, Righ_flank and Kanamycin_resistance with Synechocystis PCC. δslr0906 as template using touchdown PCR.
  2. Gel electrophoresis 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 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.

  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. Gel electrophoresis 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. Gel electrophoresis 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, heat killed at 80 °C for 30 minutes, then stored at -20 °C.

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

Week 26

(23/06 - 29/06)

June 23rd
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Ligation was performed at 42 °C for 20 minutes using Taq 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 E. coli DH5α at 45 °C and plated out on LB plates 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 Taq ligase instead of T4 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 3A assembly 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 Taq 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.

Ligation and heat-shock transformation of digestion mixtures from June 20th.

June 24th
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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 E. coli DH5α cells; however, ligation still failed.
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.

  1. Attempted ligation and heat-shock transformation of the digestion mixtures from June 20th again.
  2. Gel electrophoresis verification of digest from June 20th on agarose gel.
  3. Inoculated new colonies of BBa_J23101, BBa_B0034 and BBa_C0012.

June 25th
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Enzymatic digestion performed with similar procedure as June 20th: upstream part (J) cut with EcoRI-HF and SpeI, downstream part (B) cut with XbaI and PstI-HF and psB1C3 backbone cut with EcoRI-HF and PstI-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.
Verification of Left_flank, Right_flank and Kanamycin_resistance on gel showed expected bands at 566 bp, 552 bp and 961 bp respectively.

  1. Mini-prep of BioBrick colonies from June 24th.
  2. Enzymatic digestion of newly mini-prepped BBa_J23101, BBa_B0034 and psB1C3 backbone.
  3. New PCR amplification of Left_flank, Righ_flank and Kanamycin_resistance using touchdown PCR.
  4. Gel electrophoresis verification of PCR products on agarose gel.

June 26th
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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 E. coli DH5α cells contain correct plasmids.
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 PstI-HF could be counteracted by increasing Taq 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 LB plates with chloramphenicol.

  1. Gel electrophoresis verification of BBa_J23101, BBa_B0034 and psB1C3 backbone backbone digest.
  2. Another attempted ligation of digested B, J and psB1C3 backbone from June 25th, and heat-shock transformation into competent E. coli DH5α cells.

June 27th
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The ligation procedure was performed as June 26th, except that the ligation time was 20 minutes. Transformation into competent E. coli DH5α cells followed standard protocol. The next day two colonies were observed on the plate.

  1. QIAquick PCR purification of Left_flank, Righ_flank and Kanamycin_resistance PCR product from June 25th.
  2. Digested and ligated Right_Flank, Kanamycin_resistance and psB1A3 backbone, then heat-shock transformed it into competent E. coli DH5α cells.
  3. Inoculated colonies from ligation of BBa_J23101, BBa_B0034 and psB1C3 backbone from June 26th.

June 28th
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Mini-prep of inoculated colonies from BBa_J23101, BBa_B0034 and psB1C3 backbone ligation on June 26th.

Week 27

(30/06 - 06/07)

June 30th
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. 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.

  1. Gel electrophoresis verification of BBa_J23101, BBa_B0034 and psB1C3 backbone ligation on agarose gel.
  2. Digestion, ligation and heat-shock transformation of the ligated JB with Left_flank and psB1A3 backbone.

July 1st
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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 EcoRI-HF, PstI-HF, SpeI and XbaI could have caused uneven cutting of the plasmids, which could have produced erroneous bands.
The agarose gel of NotI-HF digested {RF, Kan, psB1A3} and {J,B, psB1A3} showed bands at around 1000 bp for all samples, which is wrong in both cases.

  1. New gel electrophoresis verification of BBa_J23101, BBa_B0034 and psB1C3 backbone ligation on agarose gel using NotI-HF restriction enzyme.
  2. Also verified Righ_flank, Kanamycin_resistance and psB1A3 backbone ligation from June 27th on agarose gel using NotI-HF.
  3. Inoculation of Left_flank, JB and psB1A3 backbone colonies from June 30th.

July 2nd
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All samples showed erroneous bands on the agarose gel, and it was therefore decided to restart the whole ligation process.

  1. Mini-prep of {LF, JB, psB1A3} from June 30th.
  2. Gel electrophoresis verification of {RF, Kan, psB1A3}, {J,B, psB1A3} and {Left_flank, JB, psB1A3} on agarose gel using NotI-HF restriction enzyme.
  3. Inoculated new colonies from plates containing colonies from the original BBa_J23101 and BBa_B0034 BioBricks.

July 3rd
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The gel showed correct band lengths for all samples. J, LF and Kan were digested using EcoRI-HF and SpeI. B and RF were digested using XbaI and PstI, while the backbones were digested using EcoRI-HF and PstI. Since it was not possible to heat-kill the high fidelity PstI 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.

  1. Mini-prep of inoculated BBa_J23101 and BBa_B0034 colonies from July 2nd.
  2. Gel electrophoresis verification of J and B on an agarose gel using NotI-HF restriction enzyme.
  3. Digested J, B, Left_flank, Righ_flank, Kanamycin_resistance, psB1A3 backbone and psB1C3 backbone.

July 4th
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Ratio between J/B and backbone in the ligation mixture was 2.5, and T4 ligase was used. Previously Taq ligase had been used, but because of poor transformation efficiency we decided to try T4 ligase. Growth was seen the next day. NanoDrop of Kan and RF products showed 84.3 and 95.7 ng/mL respectively.

  1. Ligated BBa_J23101, BBa_B0034 and psB1C3 backbone, then heat-shock transformed plasmid into competent E. coli DH5α.
  2. Amplified Kanamycin_resistance and Righ_flank using touchdown PCR.

July 5th
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Picked and inoculated one colony from BBa_J23101, BBa_B0034 and psB1C3 backbone ligation from July 4th.

July 6th
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No colonies were seen on the LB plates containing presumed ligated Kan and RF. New colonies of J,B and ps1C3 backbone were picked because the inoculated mixture from July 5th was blank, indicating that there had been no growth.

  1. Ligated Kanamycin_resistance, Righ_flank and psB1A3 backbone, then heat-shock transformed plasmid into competent E. coli DH5α.
  2. Picked and inoculated three colonies from BBa_J23101, BBa_B0034 and psB1C3 backbone ligation from July 4th.

Week 28

(07/07 - 13/07)

July 7th
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NanoDrop of amplified backbone showed an average concentration of 50 ng/mL for all samples. The ligation ratio of insert and backbone was 3. T4 ligase was used. The next day colonies were observed for both ligations.

  1. Amplification of psB1A3 backbone, psB1C3 backbone and psB1K3 backbone using touchdown PCR.
  2. Digestion, ligation and heat-shock transformed of Left_flank, JB with psB1A3 backbone and Kanamycin_resistance, Righ_flank with psB1A3 backbone.

July 8th
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Construct was optimised to work in Synechocystis sp. PCC 6803.

  1. Made LB plates with ampicillin and with chloramphenicol.
  2. Heat-shock transformed synthesised promoter + RBS region (Construct) into competent E. coli DH5α.
  3. One colony from each ligation on July 7th were picked and inoculated.

July 9th
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NanoDrop of LF+JB and Kan+RF showed a concentration of 44.7 and 47.6 ng/mL respectively. BBa_E1010 was picked from plate 3, 11N.

  1. Mini-prep of inoculated colonies of Left_flank+JB and Kanamycin_resistance+Righ_flank.
  2. Digestion, ligation and heat-shock transformation of LFJB, BBa_C0012 and psB1C3 backbone.
  3. Rehydrated BioBrick BBa_E1010 and heat-shock transformed into competent E. coli DH5α.

July 10th
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The sequencing results arrived approximately a week later with news that the sequence that was submitted did not correspond to the desired sequence. The next day BBa_E1010 and KRF did not show growth in the liquid medium.

  1. Gel electrophoresis verification of digest of Left_flank+JB, BBa_C0012, BBa_E1010 and psB1C3 backbone from July 9th on agarose gel using NotI-HF restriction enzyme.
  2. LFJB on psB1A3 backbone and KRF on psB1A3 backbone sent for sequencing.
  3. Inoculated new colonies of LFJB and KRF and BBa_E1010 because the gel verification did not give expected bands.

July 11th
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NanoDrop of Construct and LFJB showed an average concentration of 103 and 70 ng/mL respectively. The gel only showed bands corresponding to the length of a backbone (~2000 bp), meaning that all the samples were incorrect.

  1. Mini-prep of Construct and Left_flank+JB samples.
  2. Gel electrophoresis verification of all samples of Construct and LFJB on agarose gel using XbaI and PstI restriction enzymes.
  3. Picked and inoculated new colonies of BBa_E1010 and KRF.

July 12th
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NanoDrop of BBa_E1010 and KRF showed an average concentration of 96.6 and 118.9 ng/mL respectively. Since PstI was being used instead of PstI-HF due to heat resistance issues with the high fidelity enzyme, the buffer used during digest had to be changed. Instead of using CutSmart buffer, Neb buffer was used for PstI and SpeI. PstI should still work in CutSmart buffer even though it would not have maximum activity; however, due to problems with digests and ligations it was decided to try and change the buffer. The gel showed no bands, and we could only conclude that the samples does not contain the correct DNA sequence.

  1. Mini-prep of BBa_E1010 and KRF solutions.
  2. Digest of E1010, Construct and psB1K3 backbone.
  3. Gel electrophoresis verification of KRF samples on agarose gel.

July 13th
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The gel still did not show expected bands for BBa_E1010 and Construct.

  1. Made new SOC medium and new LB plates with kanamycin.
  2. Another gel electrophoresis verification attempt with the digests from July 12th.

Week 29

(14/07 - 20/07)

July 14th
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Tested XbaI, PstI, PstI-HF, EcoRI-HF, SpeI and NotI-HF. All enzymes cut the plasmid properly, and gave expected bands on the gel. This means that there is nothing wrong with the enzymatic activity.
Used a ratio of 1 in the ligation of insert and backbone.

  1. Gel electrophoresis verification of restriction enzymes.
  2. Attempted a new digestion, ligation and heat-shock transformed of Kanamycin_resistance, Righ_flank and psB1A3 backbone.

July 15th
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NanoDrop after PCR amplification of LF, RF and Kan showed concentration of 54.4, 113 and 69.6 ng/mL respectively. NanoDrop measurements after the digest showed concentrations reaching zero for all samples. It was later realised that wrong combination of enzymes had been used, which neatly explains the puzzling results. The amounts of enzyme and DNA was determined according to enzymatic activity in relation to weight of DNA present.
BBa_K576005 was found in plate 1, well 10A. BBa_K576005 equals J and B BioBrick ligated together. We decided to use this BioBrick instead of J and B separately because of all the trouble we were having.

  1. Gel electrophoresis verification of Left_flank, Righ_flank and Kanamycin_resistance using touchdown PCR.
  2. Digested RF, Kan and psB1A3 backbone.
  3. Rehydrated BioBrick BBa_K576005 and heat-shock transformed into competent E. coli DH5α.

July 16th
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. NanoDrop of LF, BBa_C0012, BBa_E1010, Kan, RF, psB1C3 backbone, BBa_K576005 and Construct showed 133.4, 14.4, 232.2, 215.7, 228.6, 170.6, 206.2 and 378 ng/mL respectively. We decided to give up on the 3A assembly method, since it obviously was not working for us. Instead we designed primers for Gibson assembly.

  1. PCR amplification of Left_flank, BBa_C0012, BBa_E1010, Kanamycin_resistance, Righ_flank, psB1C3 backbone, BBa_K576005 and Construct for Gibson assembly using designed primers.
  2. Made a new batch of competent E. coli DH5α cells.

July 17th
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The previous attempt resulted in very low concentration of BBa_C0012. This time around three different templates were used; (1) from the initial Mini-Prep from June 25th; (2) from the initial LB plate from June 16th; and (3) DNA from the initial PCR from June 18th. NanoDrop of BBa_C0012 samples were as following; (1) 34.5 ng/mL; (2) 6.3 ng/mL; and (3) 88.4 ng/mL. The gel showed correct bands for all samples apart from the psB1K3 backbone.

  1. PCR amplification of BBa_C0012 for Gibson assembly using designed primers.
  2. Made two batches of LB plates with kanamycin + chloramphenicol + IPTG.
  3. Gel electrophoresis verification the DNA fragments from July 16th and July 17th on agarose gel.

Week 32

(04/08 - 10/08)

August 4th
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  1. Inoculation of Gibson product assembled with partial ligation in 5 ml SOC with kanamycin, chloramphenicol and IPTG. Incubated at 37 degrees Celsius overnight.
  2. The Gibson partial ligation was also plated out on plates containing kanamycin, chloramphenicol and IPTG.

August 5th
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  1. Mini-prep of inoculation from yesterday with the Gibson partial ligation. Concentration measured at 67.6 ng/µl with nanodrop.
  2. OD730 of Synechocystis culture was determined to be 0.346. Culture was concentrated to OD730: 2.5 before transforming with the 15 minutes Gibson PCR product.
    3. /i>I-HF due to heat resistance issues with the high fidelity enzyme, the buffer used during digest had to be changed. Instead of using CutSmart buffer, Neb buffer was used for

August 6th
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  1. The Gibson partial ligation product was amplified with PCR with phusion enzyme.
  2. The amplified Gibson partial ligation product was purified with PCR purification kit.
  3. The purified Gibson partial ligation product was analyzed with gel electrophoresis.

Week 37

(08/09 - 14/09)

September 12th
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    PCR amplification of LF (left flank), RF (right flank) and kan (kanamycin) from Synechocystis with kanamycin resistance and pSB1C3 from biobrick E1010.

Week 38

(15/09 - 21/09)

September 16th
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    Verification of LF, RF, Kan and pSB1C3 was done with gel electrophoresis. Samples were purified with PCR purification kit. LF, RF, Kan, pSB1C3, GOx (glucose oxidase), Lac (lac operator) was digested with EcoRI-HF and PstI-HF for 90 minutes at 37 degrees Celsius. Digests purified with PCR purification kit and stored in freezer.

September 17th
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    Concentration of LF, RF, Kan, pSB1C3, GOx and Lac was measured with nanodrop, and amplified with PCR. Two parallels of LF, RF and Kan (no. 1 and no.2) were run. PCR products were verified with gel electrophoresis and purified with PCR purification kit. Purified products stored in freezer.

September 18th
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    Digestion of LF, RF, Kan, pSB1C3, GOx, Syn: LF1, RF1, Kan, pSB1C3, GOx, Syn with EcoRI and PstI, LF2 with EcoRI and SpeI, RF2 and Kan with XbaI and PstI. Samples inoculated at 37 degrees Celsius for 90 minutes, and thereafter purified with PCR purification kit. Concentrations were measured with nanodrop. The following digests were ligated with T4-ligase: pSB1C3 + LF1 (1:3), pSB1C3 + RF1 (1:3), pSB1C3 + Kan1 (1:3), pSB1C3 + GOx (1:3), pSB1C3 + Syn (1:5), pSB1C3 + LF2 + Kan2 (1:3:3). Ligation samples left in freezer overnight.

September 19th
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    Transformed yesterday’s ligations into E. coli DH5α cells. Inoculated overnight at 37 degrees Celsius.

Week 39

(22/09 - 28/09)

September 22nd
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    Two colonies were picked from LF1, RF1, Kan1, GOx, Syn, LF2+Kan2 and used for colony PCR. Colonies of Kan1 or LF2+Kan2 were inoculated in 4 ml SOC containing 1µg/ml chloramphenicol and kanamycin, while the remaining samples were inoculated in 4 ml SOC containing 1µg/ml chloramphenicol. PCR-products were verified with gel electrophoresis.

September 23rd
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    Mini-prep of LF1, RF2, Kan1, GOx1, Syn1, LF+Kan1. Digest of 2x LF2+Kan2 with EcoRI and SpeI. Digests was cleaned with PCR cleanup, and ligated overnight with RF2 and pSB1C3 using T4-ligase.

September 24th
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    Transformed 2x of pSB1C3, LF, Kan and RF into E. coli DH5α. Incubated at 37⁰C overnight.

Week 40

(29/09 - 05/10)

September 25th
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    Four colonies from two of yesterdays plates were selected, and inoculated in 4 ml SOC with kanamycin. The same colonies were also used for colony PCR. PCR-products verified with gel electrophoresis.

September 26nd
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    Mini-prep of 2 parallels of LF+Kan+RF+pSB2C3 (ligation). Digested with EcoRI and NotI. Samples run on gel, one sample verified and selected for Biobrick shipping.

October 1st
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    Concentration of miniprepped biobricks LF (24.8 ng/µl), RF (33.3 ng/µl), Kan (40.9 ng/µl), GOx (30.1 ng/µl), Lac (34.6 ng/µl) and Kan + RF (39.8 ng/µl) were measured with nanodrop.

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