Team:NTNU Trondheim/Notebook

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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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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 PCC. δslr0906 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 posli id="pt-login">sible 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 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 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.

Week 39

(22/09 - 28/09)

September 22nd
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  1. 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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  1. 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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  1. Transformed 2x of pSB1C3, LF, Kan and RF into E. coli DH5α. Incubated at 37⁰C overnight.

Week 40

(29/09 - 05/10)

October 1st
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  1. 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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