Team:TU Darmstadt/Notebook/LJ-Yeast

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Production of glycolic acid in S. cerevisiae

July
Week 1 (2^nd of July – 8^th of July)
We finally started with labwork by performing a PCR with the AtGLYR1 sequence. Unfortunately an agarose gel electrophoresis showed that the PCR was not successful.♣
Week 2 (9^th of July – 15^th of July)
We repeated the PCR from last week and performed another PCR for the plasmid pAT423 containing GLYR1. An agarose gel electrophoresis showed a few positive samples.
Week 3 (16^th of July – 22^nd of July)
The positive samples were purified. However, we performed another PCR with both plasmids to see if a different master mix and PCR cycle would have an impact on our output. An agarose gel electrophoresis showed only one positive result. So we repeated it, but this time with different annealing temperatures and a different GC-Enhancer. One positive result was screened via an agarose gel electrophoresis and purified afterwards.
Week 4 (23^rd of July – 29^th of July)
With the positive approach from last week we performed a new PCR. The following agarose gel electrophoresis confirmed the success of this PCR. The samples got purified afterwards. To check these positives results again we repeated the successful PCR with the plasmid pAT423 containing GLYR1 and performed a restriction digest. An agaorse gel showed that all fragments had the expected length.
Besides, we started with the work on our second gene icl1. by performing a Gibson Assembly, which was followed by a PCR. Three templates had the right length, which was veriefied via an agarose gel electrophoresis. Those got purified and were used for another PCR.

August
Week 5 (30^th of July – 5^th of August)
In this week we performed several PCRs with icl1. I think after this year we will definitely become a master in performing PCRs. Alongside to this, we performed a restriction digest with pSB1C3 and the PCR products of glyr1 and icl1. The digestion product was ligated into a dephosphorylated pSB1C3 backbone. The ligation product was transformed into E. coli. At the end a colony PCR was performed.
Week 6 (6^th of August – 12^th of August)
First, a linearization of GlyR1 x pAT423-plasmid was made via another PCR approach. For ICL1 we repeated the PCR, which showed positive results on an agarose gel. GLYR1 and ICL were prepared and a colony PCR was performed. An agarose gel electrophoresis revealed, that only 2 templates for GLYR1 have worked. In this week we started with the planned deletion of MLS1 and IDP2. For MLS1, deleted through a PCR-amplified KanMX-cassette and for IDP2 via a Leucin-cassette. A PCR clean-up was performed and MLS1 was transformed into competent yeast cells for the IDP2 deletion.
Week 7 (13^th of August – 19^th of August)
We performed a new colony PCR for ICL1. Due the fact, that the cloning with ICL1 does not work properly, we amplified ICL1 with longer overhangs. After the positive PCR of GLYR1 with the pSB1C3 backbone we prepared the samples and send them for DNA sequencing. This we did as well for ICL1 after the cPCR this week was successful. After the results of ICL1 were positive, we created glycerol-stocks. To proof whether the Leucin-cassette for the deletion had worked, we disrupted the cells and performed a cPCR.
Week 8 (20^th of August – 26^th of August)
This week we wanted to purify our GLYR1 via ÄKTA pure system. Unfortunately this did not work properly, which could be analyzed via an SDS-PAGE. We repeated the cell disruption and cPCR of last week and screened it via an agarose gel electrophoresis. Positive samples were prepared and a transformation of GLYR1 x pAT423 into deletion of MLS1/IDP2 was made.

September
Week 9 (27^th of August – 2^nd of September)
We performed a new miniprep for GLYR1 x pSB1C3 and sent it for sequencing. While we were waiting for the results of the sequencing we performed a retransformation of GLYR1 xpsB1C3 and a restriction digest.
Week 10 (3^rd of September – 9^th of September)
We analyzed GLYR1 x pAT423 with and without the different deletion via different SDS-PAGES and one western blot.
Week 11 (10^th of September – 16^th of September)
This week we repeated the SDS-PAGE and western blot to verify our results.
Week 12 (17^th of September – 23^rd of September)
A new protein purification with GLYR1 x pAt423 was performed this week with other settings than last time. And it succeeded, which we proved via an SDS-PAGE this week. Besides, we worked again with GLYR1 x pSB1C3 with new overhangs and transformed the ligation product into E. coli TOP 10.
Week 13 (24^th of September – 30^th of September)
To prove our purification was positive, we performed a western blot with the purified GlYR1 x pAT423, which succeeded. For GLYR1 x pSB1C3 we performed a cPCR and sent the positive samples for sequencing. We started with our enzyme assays this week as well. For this, we first disrupted the cells. For the GLYR1 x pAT423 we performed a NADPH-dependent assay, which we repeated to verify our positive results. Furthermore we made a new western blot. For GLYR1 x pSB1C3 we started a new attempt for cloning.
October
Week 14 (1^st of October – 7^th of October)
We performed a restriction digest for GLYR1 x pSB1C3 out of a plasmid prep, which we did before. Positive colonies were screened via agarose gel electrophoresis and sent in for sequencing. And all samples were positive. Now we have GLYR1 correct in pSB1C3. Subsequently, we created glycerol stock. Besides, we performed two more assays this week, a HPLC and a growth curve.
Week 15 (8^th of October – 14^th of October)
This week we just repeated the growth curve assay again. And we are totally finished in the lab.
Week 16 (15^th of October – 21^st of October)
… and cleaning of the Lab
Week 17 (22^nd of October – 28^th of October)
Fly to Boston and attend the Giant Jamboree!