Team:Technion Israel/Lab Notebook/Overview
Lab Notebook Overview
Week 1: May 2-8
Bacillus
We designed gblocks for our gene (3-α-HSD): one for the original sequence and one Bacillus-optimized.
Active team members this week: Liron, Tal, and Ruth
E.coli
Planned the Gibson reaction for our gBlock of 3α-HSD and the plasmid N155 (from our mentors) containing pT7, RBS and mcherry.
Ordered the gBlock of 3α-HSD, which was optimized for E. coli codon usage. We also ordered primers for the Gibson reaction for the gBlock.
Performed reverse PCR to the N155 plasmid and carried out a DPN1 reaction on the product.
Active team members this week: Adi and Alexey
Modeling
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Comb
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NADPH
This week we did a lot of research. We found that the 3-α-HSD enzyme can use both NADH and NADPH as co-factors for the reduction reaction in order to inactivate DHT to 3-α-diol.
NADPH has been found to not only enable the reaction in the direction we want, but also to inhibit the reverse reaction if DHT (Rizner, et al.) Therefore, we have decided to try to overproduce NADPH in a host organism, in order to support the activity of the enzyme.
Active team members this week: Yael and Roni
Week 2: May 9-15
Bacillus
We received the gblocks and performed PCR. We had some difficulties amplifying the Bacillus-optimized gblock, and since we found out that B.subtilis has no preferred codon usage, we decided to continue only with the gblock of the original sequence.
Active team members this week: Liron, Tal, and Ruth
E.coli
Our gBlock arrived!!!
We performed PCR on the 3αHSD gBlock with only 10 cycles (as recommended), but received no band in the gel
We performed PCR on the 3αHSD gBlock in higher temperatures, but still received no bands in the gel
Active team members this week: Adi
Modeling
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Comb
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NADPH
We conducted further research about the production of NADPH. We sent e-mails to authors of journal articles to enquire about genes and knockouts associated with NADPH overproduction.
Active team members this week: Yael and Roni
Week 3: May 16-22
Bacillus
We performed a PCR reaction for the mCherry reporter gene from pUG34 plasmid we got from Inbal Vaknin from Prof. Roee Amit’s lab in the Technion and added a RBS sequence with the primers.
We cleaned the product and performed a restriction enzyme reaction with KpnI and HindIII.
Active team members this week: Liron, Tal, and Ruth
E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
Received replies from various authors. Prof. Toby Fuhrer, from the Institute of Molecular Systems Biology will be sending us an E. Coli MG1655 strain with a knockout of pgi and UdhA genes associated with the consumption of NADPH. Prof. Hanna Engleberg-Kulka’s lab at the Hebrew University of Jerusalem will be sending us a plasmid containing a zwf gene, which codes for the Glucose-6-Phosphate Dehydrogenase enzyme related to the Pentose Phosphate pathway, in which NADPH is produced.
Active team members this week: Yael and Roni
Week 4: May 23-29
E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
We received the knockout strain from Prof. Fuhrer! We prepared glycerol stock and competent cells of the strain for further use.
Planned the PCR and ordered primers for the zwf gene containing the sequence for cutting sites for SpeI and XbaI to make ligation with biobricks simple.
Active team members this week: Yael and Roni
Week 5: May 30-June 5
Bacillus
We met with Shira Omer from Avigdor Eldar’s lab from Tel Aviv University and Prof. Ilana Kolodkin from Weizmann Institute. They both recommended working with pDR111 plasmid (a shuttle vector which integrates into the Bacillus genome).
Active team members this week: Liron, Adi, Yael, and Ruth
E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
We are still waiting to receive the plasmid containing the zwf gene. Apparently there are delays in the postal service. Since we are scared the DNA will break down, we have hired a messenger service to bring it to us early next week.
Planned activity verification experiments for when we have our clones. We will do preliminary experiments using fluorescence, which will indicate total NADPH and NADH levels, and then use the Biovision NADPH/NADP+ Assay kit to verify our results.
Active team members this week: Yael and Roni
Week 6: June 6-12
Bacillus
We checked the plasmids we got from TAU and Weizmann Institute by sending for sequencing.
Active team members this week: Liron, Tal, and Ruth
E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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Week 7: June 13-19
Bacillus
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E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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Week 8: June 20-26
Bacillus
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E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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Week 9: June 27-July 3
Bacillus
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E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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Week 10: July 4-10
Bacillus
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E.coli
We took a break for our exams.
Modeling
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Comb
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NADPH
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Week 11: July 11-17
Bacillus
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E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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Week 12: July 18-24
Bacillus
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E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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Week 13: July 25-31
Bacillus
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E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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Week 14: August 1-7
Bacillus
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E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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Week 15: August 8-14
Bacillus
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E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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Week 16: August 15-21
Bacillus
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E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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Week 17: August 22-28
Bacillus
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E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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Week 18: August 29-September 4
Bacillus
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E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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Week 19: September 5-11
Bacillus
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E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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Week 20: September 12-18
Bacillus
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E.coli
Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
Modeling
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Comb
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NADPH
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