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Revision as of 19:27, 1 September 2015
Lab Notebook Overview
Week 1: May 2-8
Bacillus
We designed gBlocks for our gene (3α-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, from IDT, 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
NADPH
This week we did a lot of research. We found that the 3α-HSD enzyme can use both NADH and NADPH as co-factors for the reduction reaction in order to inactivate DHT to 3α-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
Modeling
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Comb
A mechanical engineering student, Michael Yannai, helped me design the first prototype of the comb. We designed it to look like a lice comb with internal channels and thick teeth. The main entrance to the channels is located at the side of the comb.
Active team members this week: Maayan
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 3α-HSD gBlock with only 10 cycles (as recommended), but received no band in the gel.
We performed PCR on the 3α-HSD gBlock in higher temperatures, but still received no bands in the gel.
Active team members this week: Adi
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
Modeling
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Comb
Met with Prof. Hovav Gazit and set a date for printing the comb.
Active team members this week: Maayan
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 3α-HSD gBlock in lower temperatures, no bands in the gel.
Active team members this week: Alexey
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
Modeling
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Comb
The first prototype of the comb was been printed in Prof. Hovav Gazit's lab with a 3-D printer. Unfortunately, the channels were blocked with ABS from the printer.
Active team members this week: Maayan and Sagi
Week 4: May 23-29
E.coli
Performed PCR to the 3α-HSD gBlock with more cycles and 15°C temperature gradient- no bands in the gel.
Active team members this week: Alexey and Adi
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
Modeling
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Comb
We tried to open the channels inside the comb with a water pressure machine in Prof. Hovav Gazit's lab and mechanically with needle. Our attempts were unsuccessful.
Active team members this week: Maayan and Sagi
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
We ordered new primers for the 3αHSD gBlock.
PCR to the 3α-HSD gBlock with new primers and 15°C temperature gradient. We saw weak bands in the gel, but found no DNA after cleaning.
Active team members this week: Alexey and Adi
NADPH
We are still waiting to receive the plasmid containing the zwf gene. Apparently there are delays in the postal service. Since we suspect that 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
Modeling
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Comb
We designed a second prototype. We changed the location of the main entrance hole to the center, top of the comb, to try to enable us to open the channels, mechanically, more easily .
Active team members this week: Maayan and Sagi
Week 6: June 6-12
Bacillus
We checked the plasmids we got from Tel Aviv University and Weizmann Institute by sending for sequencing.
Active team members this week: Liron, Tal, and Ruth
E.coli
We performed a PCR reaction again with the new primers. We saw no bands in the gel.
We planned a new strategy for cloning using restriction enzymes, with BBa_K784023 in pSB1C3 (from iGEM12_Technion) as the backbone.
Active team members this week: Alexey and Adi
NADPH
We received the plasmid carrying the zwf gene and did PCR to enhance the gene. We cut biobrick BBa_K525998- pSB1C3 with a pT7 promoter and RBS, with SpeI and ligated with our gene.
Transformation of the biobrick into Top10.
Colony PCR to the colonies showed three positive colonies. We sent one for sequencing.
Active team members this week: Yael and Roni
Modeling
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Comb
We printed the second prototype of the comb. The flow that came out of the teeth of the comb was not simultaneous.
Active team members this week: Maayan and Sagi
Week 7: June 13-19
Bacillus
We designed primers for the 3α-HSD gBlock and mCherry, containing the sequenced of the new restriction enzymes (SalI and NheI).
Active team members this week: Liron, Tal, and Ruth
E.coli
Made starters from Top10 with BBa_K784023 from glycerol stock, and the performed a miniprep for the BBa_K784023. The DNA concentrations were low, so we repeated the miniprep. After receiving a satisfactory concentration, we cut the plasmid with SpeI and XbaI and performed a CIP reaction.
Ordered new primers for both Gibson and restriction enzymes cloning and performed PCR to the 3α-HSD gBlock with all the new primers. We saw a band in the negative control.
Active team members this week: Adi
NADPH
The sequencing results for the plasmid carrying our gene came out negative ☹. But we didn't give up! We performed colony PCR again, and this time saw one positive colony, which we sent for sequencing.
We transformed the gene as it was received on the PQE-32 plasmid with a pT5 promoter and the LacIq operon into the E. Coli MG1655 knockout and E. Coli BL21
.Active team members this week: Yael and Roni
Modeling
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Comb
We searched for a microfluidics flow specialist and set up a meeting with Prof. Moran Bercovici from mechanical engineering faculty at Technion.
Active team members this week: Maayan and Sagi
Week 8: June 20-26
E.coli
Performed PCR to the 3α-HSD gBlock with all the new primers and saw weak bands in the gel, but no DNA after cleaning
Active team members this week: Adi
NADPH
The sequencing results for the gene on the pSB1C3 came out positive!! We transformed the plasmid into E. Coli BL21 and the E. Coli MG1655 knockout. Unfortunately no colonies were formed on the LB and agar plates, so we repeated the transformation, but to no avail.
We received a plasmid carrying the Rhl-R inducible operon from Prof. Roee Amit’s lab. We will be using it to compare inducible NADPH overproduction to constitutive production.
Active team members this week: Yael and Roni
Modeling
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Comb
Meeting with Prof. Moran Bercovici, a specialist in microfluidic flow. He gave us a lot of information about the flow inside the channels.
Active team members this week: Maayan and Sagi
Week 9: June 27-July 3
Bacillus
We performed a PCR reaction to amplify the mCherry with the new restriction sites. We also performed a PCR reaction to amplify the 3α-HSD gBlock. We cleaned these reactions and cut them both with SalI and NheI restriction enzymes and cleaned again.
Active team members this week: Ruth
E.coli
Performed PCR to the 3α-HSD gBlock with all the new primers but saw no bands in the gel. Therefore, we decided to try a new direction to stop using our gBlock and try using the gBlock of 3α-HSD with the original (non-optimized) sequence.
We ordered new primers for the gBlock of 3α-HSD with the original sequence. We planned them based on cloning with restriction enzymes.
Active team members this week: Alexey and Adi
NADPH
Repeated transformation of the pSB1C3 plasmid containing the zwf gene, this time using newly prepared plates and heat shock instead of electroporation. The colonies grew! We prepared glycerol stocks of each strain.
Ordered primers containing KpnI and XbaI restriction sites for cloning the zwf gene into the plasmid containing the Rhl-R operon.
Active team members this week: Yael and Roni
Modeling
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Comb
We designed a new version of the comb, changing the channel structure based on the advice we received during the meeting with Prof. Moran Bercovici.
We also added a handle to the comb for more comfortable use, a larger place for the syringe, and thinner teeth for better brushing.
Active team members this week: Maayan and Sagi
Week 10: July 4-10
Bacillus
We cut the pDR111 plasmid we received from Tel Aviv University with restriction enzymes SalI and NheI. We sent it to sequencing.
Meanwhile, after cleaning from the restriction enzyme reaction, we performed a CIP reaction and cleaned. We performed a ligation reaction with the cleaned plasmid and pre-cut mCherry and 3α-HSD gBlock.
We did a heat shock transformation into competent E. Coli DH5α we got from Prof. Ilana Kolodkin Gal’s laboratory at Weizmann Institute.
We performed a colony PCR for both transformations: we got 3 positive colonies out of 9 for the pDR111 and mCherry reaction, but no positive colonies ifor the pDR111 and 3α-HSD gBlock reaction (out of 9). Therefore, we repeated the ligation step, but this time incubated overnight with a self-ligation control. We also made a starter for one of these colonies for glycerol stock and miniprep.
We transformed the pDR111 and mCherry into B.subtilis PY79 according to a protocol we got from Tel Aviv University.
We also ordered a new gVlock for the 3α-HSD gene with a signal peptide and with adaptations to iGEM demands for biobricks.
We performed PCR for the 3α-HSD gBlock containing the sequence for signal peptide aprE. After cleaning, there was a very low concentration.
Active team members this week: Ruth and Tal
NADPH
We performed PCR to the zwf gene using the primers planned specifically for ligation into the Rhl-R-containing plasmid.
Restriction and ligation of the zwf PCR product and the Rhl-R-containing plasmid.
Modeling
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Comb
We printed the new design in Prof. Moran Bercovici's lab. Some of the channels were still blocked, but the flow from the comb teeth was simultaneous. However, the handle was too small to be used comfortably by those with large hands.
Active team members this week: Maayan and Sagi
Week 11: July 11-17
E.coli
Performed PCR on the 3α-HSD gBlock with the original sequence (RFC10 compatible), with new primers and a 15°C temperature gradient. We saw good bands on gel.
Active team members this week: Alexey and Adi
NADPH
Transformation of the zwf PCR product and the Rh1-R-containing plasmid into of the Top10.
Two positive colonies identified by colony PCR were sent for sequencing.
Modeling
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Comb
We designed two containers to fit in the comb to hold our solutions containing Bacillus Subtilis (which secretes the 3α-HSD enzyme) and E. Coli (designed to overproduce NADPH).
We designed the handle and the body of the comb to be printed separately because of the enlargement of the handle was too big to be printed by the 3-D printer in one piece.
Active team members this week: Maayan and Sagi
Week 12: July 18-24
Bacillus
We did a colony PCR to B.subtilis containing mCherry and E. Coli DH5a with the 3α-HSD gBlock.
We made glycerol-stock and performed a miniprep for the positive colony of E. Coli DH5a with the 3α-HSD gBlock on the PDR111 plasmid.
We transformed the pDR111 plasmid containing the 3α-HSD gBlock into B.subtilis PY79 and performed a colony PCR.
We repeated the PCR for the signal peptide (SP) and 3α-HSD gBlock with less primer (1ul each). This time the PCR worked so we cleaned it and cut it with SalI and NheI restriction enzymes. We then performed an overnight ligation of the cut SP and 3α-HSD gBlock with pre-cut pDR111 plasmid. We transformed the product into E. Coli TOP10 and performed a colony PCR. Starters for positive colonies (4 and 9) were made, from which we prepared glycerol-stock and performed a miniprep.
Active team members this week: Ruth, Liron, and Tal
E.coli
We performed a restriction reaction of the 3α-HSD gBlock with the original sequence (RFC10 compatible)using SpeI and XbaI enzymes. We then ligated the cut 3α-HSD with pSB1C3 and transformed the ligation product into E. Coli Top10. Colonies grew in the control (ligation of pSB1C3 without 3α-HSD). Therefore, we performed a colony PCR with 8 colonies, but received no positive colonies. We repeated the colony PCR, this time with 20 colonies, but received no positive colonies.
We planned the expression plasmid with the part BBa_K1321338, which contains pT7 and an RBS (iGEM14_Imperial), and reporter plasmids with the parts BBa_J06702, containing an RBS, the mcherry gene, and a terminator (iGEM2005), and BBa_K1357008, containing an RBS and tsPurple and a terminator (iGEM14_Virginia). We then extracted the biobricks from the 2015 DNA Distribution Kit and transformed them into E. Coli Top10. We made starters from the transformed strains and performed a miniprep.
We performed a restriction reaction of BBa_K1321338 with PstI and SpeI enzymes and of BBa_J06702, BBa_K1357008 with PstI and XbaI enzymes.
We performed PCR again to the 3α-HSD gBlock with the original sequence (RFC10 compatible) and cut the 3α-HSD with with SpeI and XbaI restriction enzymes. We then repeated the ligation reaction of the 3α-HSD and the pSB1C3 plasmid and transformed the ligation product to E. Coli Top10.
After colony PCR of 3α-HSD on the pSB1C3 plasmid with 13 colonies, we received 10 positive colonies!! We sent them for sequencing, but received negative results.
Active team members this week: Alexey and Adi
NADPH
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Modeling
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Comb
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Week 13: July 25-31
Bacillus
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E.coli
Due to last week's negative results, we decided to plan a new restriction strategy for BBa_K1321338 with 3α-HSD. We performed a restriction reaction of BBa_K1321338 with SpeI only, and followed it by a CIP reaction. We then ligated the BBa_K1321338 with 3α-HSD and transformed the ligation product into E. Coli Top10. The colony PCR with 26 colonies resulted in 1 positive colony which we sent for sequencing. We finally got a positive clone!!
Active team members this week: Alexey and Adi
NADPH
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Modeling
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Comb
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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
NADPH
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Modeling
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Comb
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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
NADPH
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Modeling
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Comb
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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
NADPH
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Modeling
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Comb
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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
NADPH
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Modeling
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Comb
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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
NADPH
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Modeling
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Comb
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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
NADPH
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Modeling
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Comb
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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
NADPH
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Modeling
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Comb
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