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Team:UNIST Korea/experiment/lab note
From 2011.igem.org
July 6 We learnt how to make Electro Competent Cell. While JaeSung and Eeseul were busy making Comp cell, Bokeun and YuLim were busy setting PCR and recovering Biobrick Parts. We made a list of the biobrick parts we would use in our experiment and we tried to recover those plasmids from the DNA kit box we obtained from iGEM.
July 7 We saw pretty colonies on our plates we made yesterday. We failed to recover all of the biobrick parts we aimed for. So, we tried to recover them once again. We also have transformed the pSIM5 plasmid carrying the lambda red system into SCS110 to perform gene knockout. Then our instructor, Vinu tried to explain us the use of lambda red system for gene knockouts. We were excited with the function of exo, gamma and beta proteins. We inoculated SCS110 pSIM5 cells for tomorrow’s experiment. Eeseul made a PCR product that is to be used to knockout our target gene.
July 8 We tried to knock out gene. SCS110 is a comparatively slow growing cell. It was funny to induce cells at 42⁰C and immediately cooling them and making electrocompetent for transformation.
July 9 We were excited to see tiny small colonies on our Kanamycin plates. We went ahead to do a colony PCR to check for the true deletion. At the end of the day, we confirmed that we have deleted our gene, EnvZ. We were so excited because now we can make our E. coli to sense light. Our instructor advised us to remove the kanamycin cassette before we proceed to check the light sensing ability.
July 11 We inoculated the SCS110 ∆envZ strains to make competent cell and transform pCP20 plasmid, which would help us to remove the kanamycin cassette.
July 12 We failed to remove the kanamycin cassette. Our instructor advised us to try it again. We tried again. Eeseul got a lot of stress in removing the kanamycin cassette. So YuLim tried to remove the kanamycin cassette.
July 13 YuLim also failed to remove the kanamycin cassette. Vinu advised us to give up this strain, as the plasmid pCP20 may not be compatible with SCS110 host strain. So we decided to use MG1655. We inoculated MG1655 pSIM5 strain to delete kanamycin cassette.
July 14 Eeseul tried to delete envZ gene in MG1655. JaeSung and Bokeun were busy writing Safety rule and Abstract which is due for tomorrow. YuLim and Vinu tried to isolate the genomic DNA from Streptococcus which they will use it to amplify the Dpn cassette. It was little hard and a long boring procedure as it is a gram-positive bacterium. But, finally they got a good yield.
July 16 We were busy learning the Vector NTI program in order to design our primers. It was little hard at the beginning but we started mastering it soon.
July 18 We were still busy designing primers for our future experiments. Primers for Gibson’s Assembly was little difficult. We took care of adding RBS and additional bases for restriction sites. Finally at the end of the day almost all of our primers were being designed. Now, our instructor will check our primers and place an order for our primers.
July 21 Our primers have come. Our instructor explained us the ways to process our primers. JaeSung went crazy about the calculation and finally Bokeun helped him fix his problem. Everybody rushed to set our PCRs. YuLim got all of her holin genes successfully. But she failed to get her Dpn PCR product. Vinu suggested that she should try a gradient PCR. So, she went ahead to set a gradient PCR.
July 22 YuLim still failed to get her PCR product. Vinu suggested Dpn digestion of the genomic DNA itself to confirm if Streptococcus DNA we had really has a Dpn gene. To our suspicion, Streptococcus DNA was digested by Dpn suggesting it has no Dpn. Vinu tried to place order for another Streptococcus strain from KCTC. Bokeun went ahead to clone his fimE gene into a plasmid pACYC.
July 23 Bokeun and JaeSung are done with their cloning, they luckily got one colony, and that had their target gene inserted. Bokeun was extremely excited with it. We transformed 3 plasmids (light receptors, fimE and GFP) into one strain. JaeSung went on to check the expression of mRNA at different temperatures. The preliminary result was promising and he was able to see some difference based on the temperature.
July 25 JaeSung went on to screen for the cells Bokeun made to find one cell, which carries all 3 plasmids. He used TECAN microplate reader to screen for the cell. Finally he got one colony that contains all 3 plasmids. And Eeseul streaked it on a plate.
July 26 The last day's plate was completely green the next day. Then we realized that TECAN is dark. So, in our cell darkness lead to fimE expression which in turn inverted all of the promoters of GFP making GFP expression constitutive. So, we learnt that we should take care of darkness when we use fimE system.
July 27-29 We went for a workshop on Climate Change. We explained the audience about iGEM and our goal. We were explaining our project for the first time to a group of people. We were excited with that. We celebrated the day with alcohol.
August 1 We deleted the envZ gene and integrated cI gene into the chromosome of MG1655 using lambda red system.
August 3 We tried to transform the light sensing biobricks and the reporter gene into the envZ deleted strain
August 10 Our strain containing the light plasmid and reporter plasmids are ready. We made a quick check of its functionality in the presence and absence of light.
August 13 We got the new Streptococcus DNA and successfully amplified the Dpn genes. Gibson’s assembly works very well.
August 16 YuLim completed cloning Holin and Dpn into a plasmid without the prmoter.
August 17 YuLim went on to put the promoters in front of Holin and Dpn. She has two kinds of promoter one is a inversion promoter, ptrc* and another is a Constitutive Promoter. She is aware that it is not as easy to clone the promoters, as it was to clone the genes. She was causing in using specific strains for each promoters. (pL promoter into ECNR2; Dpn into SCS110).
September 4 Eeseul repeated the mRNA-GFP. However, she had difficulty in understanding the GFP expression as she made a temperature shift at the early log phase. She had to deal with a slow growing and fast growing strain. So, she gave up the experiment as she could not predict her output and even the control was not as expected. Bokeun tried to check the effect of different concentration of glucose on the light receptor. He could see a good cross talk of the light and the osmo-regulator.
September 5 JaeSung repeated the mRNA experiment even when his control gave good result. He could not see any difference in GFP expression with temperature dependent riboswitch.
September 7 JaeSung tried to check the effect of light on cI expression by following GFP expression every one hour. After one day of tiresome experiment, he got a very good result.
September 8 We had a deep discussion on Synthetic Biology and iGEM to Science Specialized High Schools in Korea and in Singapore.
September 11 Eeseul tried to check the effect of cross-talk between the osmo-regulator and the light receptor using salt. She found no cross-talk with salt. YuLim was almost done with her cloning of Holin and Dpn cassette. Now she is ready to check their functionality.
September 18 Bokeun and Eeseul tried to prove the cross-talk using various sugars such as Arabinose and Glucose. They found a similar pattern even when they used arabinose or glucose.
September 21 We had a group presentation with all members of our department. It was our first official presentation. We were excited and we got many good feed backs.
September 22 We were busy making our websites and documenting the results. We were aware that the wiki would freeze soon.
September 23 We went on to clone Holin and Dpn under the control of pBAD promoter as the light promoter did not work
September 25 We cloned the riboswitch regulated GFP into pTrc promoter to check for a significant change in GFP with temperature. We still could not see any significant changes in GFP.
September 28 Our instructor Vinu suggested that some promoters will not function at low temperature so trying the mRNA at 25 and 16 degree might be because of the promoter itself. So we tried to induce at 30 degree and 27 degree. Now, we got the expected result we were happy that we can give one result for iGEM. Our mRNA is more good than other cold inducible promoter systems.
