Revision as of 13:19, 2 September 2015

Team: Technion 2015

Lab Notebook Overview

Week 1: May 2-8

Secretion

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 Cofactor

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

Secretion

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

Expression

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 Cofactor

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

Secretion

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

Expression

Performed PCR on the 3α-HSD gBlock in lower temperatures, no bands in the gel.

Active team members this week: Alexey

NADPH Cofactor

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

Expression

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 Cofactor

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

Secretion

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

Expression

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 Cofactor

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

Secretion

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

Expression

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 Cofactor

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

Secretion

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

Expression

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 Cofactor

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

Expression

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 Cofactor

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

Secretion

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

Expression

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 Cofactor

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

Secretion

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 Cofactor

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

Expression

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 Cofactor

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

Secretion

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

Expression

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 Cofactor

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Modeling

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Comb

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Week 13: July 25-31

Secretion

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Expression

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 Cofactor

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Modeling

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Comb

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Week 14: August 1-7

Secretion

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Expression

Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.

Active team members this week: Alexey

NADPH Cofactor

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Modeling

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Comb

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Week 15: August 8-14

Secretion

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Expression

Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.

Active team members this week: Alexey

NADPH Cofactor

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Modeling

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Comb

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Week 16: August 15-21

Secretion

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Expression

Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.

Active team members this week: Alexey

NADPH Cofactor

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Modeling

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Comb

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Week 17: August 22-28

Secretion

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Expression

Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.

Active team members this week: Alexey

NADPH Cofactor

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Modeling

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Comb

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Week 18: August 29-September 4

Secretion

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Expression

Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.

Active team members this week: Alexey

NADPH Cofactor

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Modeling

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Comb

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Week 19: September 5-11

Secretion

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Expression

Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.

Active team members this week: Alexey

NADPH Cofactor

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Modeling

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Comb

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Week 20: September 12-18

Secretion

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Expression

Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.

Active team members this week: Alexey

NADPH Cofactor

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Modeling

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Comb

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Contact Us

@@ Line 44: / Line 44: @@
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 body {
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 				<span class="btn-bg1"></span>
 				<div class="sub1">
-					<a href="https://2015.igem.org/Team:Technion_Israel/Project/Overview">Overview</a>
+					<a href="https://2015.igem.org/Team:Technion_Israel/Project">Overview</a>
 					<a href="https://2015.igem.org/Team:Technion_Israel/Project/Bacillus">Bacillus</a>
 					<a href="https://2015.igem.org/Team:Technion_Israel/Project/E_Coli">E.Coli</a>
@@ Line 138: / Line 134: @@
 				<span class="btn-bg4"></span>
 				<div class="sub4">
-					<a href="https://2015.igem.org/Team:Technion_Israel/Practices/Education">Education</a>
+					<a href="https://2015.igem.org/Team:Technion_Israel/Practices">Practices</a>
-					<a href="https://2015.igem.org/Team:Technion_Israel/Practices/Publications">Publications</a>
+					<a href="https://2015.igem.org/Team:Technion_Israel/Practices/Safety">Safety</a>
-					<a href="https://2015.igem.org/Team:Technion_Israel/Practices/Market_Research">Market&nbsp;Research</a>
 				</div>
 			</div>
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 					<a href="https://2015.igem.org/Team:Technion_Israel/Collaborations">Collaborations</a>
 					<a href="https://2015.igem.org/Team:Technion_Israel/Design">Design</a>
-					<a href="https://2015.igem.org/Team:Technion_Israel/Entrepreneurship">Entrepreneurship</a>
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 			</div>
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 			<div class="week_content">
 				<div class="bacillus">
-					<h2><i>Bacillus</i></h2>
+					<h2>Secretion</h2>
 					<p>We designed gBlocks for our gene (3α-HSD): one for the original sequence and one Bacillus-optimized.</p>
 					<p><i>Active team members this week: Liron, Tal, and Ruth</i></p>
@@ Line 178: / Line 172: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>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.</p>
 					<p>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.</p>
@@ Line 199: / Line 193: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2><i>Bacillus</i></h2>
+					<h2>Secretion</h2>
 					<p>We received the gBlocks and performed PCR. We had some difficulties amplifying the Bacillus-optimized gblock, and since we found out that <i>B.subtilis</i> has no preferred codon usage, we decided to continue only with the gblock of the original sequence.</p>
 					<p><i>Active team members this week: Liron, Tal, and Ruth</i></p>
 				</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>Our gBlock arrived!!!</p>
                      <p>We performed PCR on the 3α-HSD gBlock with only 10 cycles (as recommended), but received no band in the gel.</p>
@@ Line 212: / Line 206: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>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.</p>
 					<p><i>Active team members this week: Yael and Roni</i></p>
@@ Line 231: / Line 225: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>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.</p>
 					<p>We cleaned the product and performed a restriction enzyme reaction with KpnI and HindIII.</p>
@@ Line 237: / Line 231: @@
 				</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>Performed PCR on the 3α-HSD gBlock in lower temperatures, no bands in the gel.</p>
                      <p><i>Active team members this week: Alexey</i></p>
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>Received replies from various authors.  Prof. Toby Fuhrer, from the Institute of Molecular Systems Biology will be sending us an <i>E. Coli MG1655</i> 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.</p>
 					<p><i>Active team members this week: Yael and Roni</i></p>
@@ Line 261: / Line 255: @@
 			<div class="week_content">
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>Performed PCR to the 3α-HSD gBlock with more cycles and 15°C temperature gradient- no bands in the gel.</p>
                      <p><i>Active team members this week: Alexey and Adi</i></p>
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>We received the knockout strain from Prof. Fuhrer! We prepared glycerol stock and competent cells of the strain for further use.</p>
 					<p>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.</p>
@@ Line 287: / Line 281: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>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 <i>Bacillus</i> genome).</p>
 					<p><i>Active team members this week: Liron, Adi, Yael, and Ruth</i></p>
 					</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>We ordered new primers for the 3αHSD gBlock.</p>
 					<p>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.</p>
@@ Line 298: / Line 292: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>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.</p>
 					<p>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.</p>
@@ Line 319: / Line 313: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>We checked the plasmids we got from Tel Aviv University and Weizmann Institute by sending for sequencing.</p>
 					<p><i>Active team members this week: Liron, Tal, and Ruth</i></p>
 				</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>We performed a PCR reaction again with the new primers.  We saw no bands in the gel.</p>
 					<p>We planned a new strategy for cloning using restriction enzymes, with BBa_K784023 in pSB1C3 (from iGEM12_Technion) as the backbone.</p>
@@ Line 330: / Line 324: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>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.  </p>
 					<p>Transformation of the biobrick into Top10. </p>
@@ Line 352: / Line 346: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>We designed primers for the 3α-HSD gBlock and mCherry, containing the sequenced of the new restriction enzymes (SalI and NheI).</p>
 					<p><i>Active team members this week: Liron, Tal, and Ruth</i></p>
 				</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>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.</p>
@@ Line 364: / Line 358: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>The sequencing results for the plasmid carrying our gene came out negative &#9785;.  But we didn't give up! We performed colony PCR again, and this time saw one positive colony, which we sent for sequencing.</p>
 					<p>We transformed the gene as it was received on the PQE-32 plasmid with a pT5 promoter and the LacIq operon into the <i>E. Coli MG1655</i> knockout and <i>E. Coli BL21</p>.
@@ Line 385: / Line 379: @@
 			<div class="week_content">
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>Performed PCR to the 3α-HSD gBlock with all the new primers and saw weak bands in the gel, but no DNA after cleaning</p>
                      <p><i>Active team members this week: Adi</i></p>
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>The sequencing results for the gene on the pSB1C3 came out positive!! We transformed the plasmid into <i>E. Coli BL21</i> and the <i>E. Coli MG1655</i> knockout.  Unfortunately no colonies were formed on the LB and agar plates, so we repeated the transformation, but to no avail.</p>
 					<p>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.</p>
@@ Line 410: / Line 404: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>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.</p>
 					<p><i>Active team members this week: Ruth</i></p>
 				</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>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.</p>
 					<p>We ordered new primers for the gBlock of 3α-HSD with the original sequence.  We planned them based on cloning with restriction enzymes.</p>
@@ Line 421: / Line 415: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>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.</p>
 					<p>Ordered primers containing KpnI and XbaI restriction sites for cloning the zwf gene into the plasmid containing the Rhl-R operon.</p>
@@ Line 442: / Line 436: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>We cut the pDR111 plasmid we received from Tel Aviv University with restriction enzymes SalI and NheI.  We sent it to sequencing.</p>
 					<p>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.</p>
@@ Line 454: / Line 448: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>We performed PCR to the zwf gene using the primers planned specifically for ligation into the Rhl-R-containing plasmid.</p>
 					<p>Restriction and ligation of the zwf PCR product and the Rhl-R-containing plasmid.</p>
@@ Line 473: / Line 467: @@
 			<div class="week_content">
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>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.</p>
                      <p><i>Active team members this week: Alexey and Adi</i></p>
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>Transformation of the zwf PCR product and the Rh1-R-containing plasmid into of the Top10.</p>
 					<p>Two positive colonies identified by colony PCR were sent for sequencing.</p>
@@ Line 498: / Line 492: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>We did a colony PCR to <i>B.subtilis</i> containing mCherry and <i>E. Coli DH5a</i> with the 3α-HSD gBlock.</p>
 					<p>We made glycerol-stock and performed a miniprep for the positive colony of <i>E. Coli DH5a</i> with the 3α-HSD gBlock on the PDR111 plasmid.</p>
@@ Line 508: / Line 502: @@
 					</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>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 <i>E. Coli Top10</i>. Colonies grew in the control (ligation of pSB1C3 without 3α-HSD).
@@ Line 522: / Line 516: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>content</p>
 				</div>
@@ Line 539: / Line 533: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>content</p>
 				</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>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
@@ Line 551: / Line 545: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>content</p>
 				</div>
@@ Line 568: / Line 562: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>content</p>
 				</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.</p>
                                          <p><i>Active team members this week: Alexey</i></p>
@@ Line 578: / Line 572: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>content</p>
 				</div>
@@ Line 595: / Line 589: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>content</p>
 				</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.</p>
                                          <p><i>Active team members this week: Alexey</i></p>
@@ Line 605: / Line 599: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>content</p>
 				</div>
@@ Line 622: / Line 616: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>content</p>
 				</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.</p>
                                          <p><i>Active team members this week: Alexey</i></p>
@@ Line 632: / Line 626: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>content</p>
 				</div>
@@ Line 649: / Line 643: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>content</p>
 				</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.</p>
                                          <p><i>Active team members this week: Alexey</i></p>
@@ Line 659: / Line 653: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>content</p>
 				</div>
@@ Line 676: / Line 670: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>content</p>
 				</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.</p>
                                          <p><i>Active team members this week: Alexey</i></p>
@@ Line 686: / Line 680: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>content</p>
 				</div>
@@ Line 703: / Line 697: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>content</p>
 				</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.</p>
                                          <p><i>Active team members this week: Alexey</i></p>
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>content</p>
 				</div>
@@ Line 729: / Line 723: @@
 			<div class="week_content">
 				<div class="bacillus">
-					<h2>Bacillus</h2>
+					<h2>Secretion</h2>
 					<p>content</p>
 				</div>
 				<div class="ecoli">
-					<h2>E.coli</h2>
+					<h2>Expression</h2>
 					<p>Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.</p>
                                          <p><i>Active team members this week: Alexey</i></p>
@@ Line 739: / Line 733: @@
 				</div>
 				<div class="nadph">
-					<h2>NADPH</h2>
+					<h2>NADPH Cofactor</h2>
 					<p>content</p>
 				</div>

Difference between revisions of "Team:Technion Israel/Lab Notebook/Overview"

Revision as of 13:19, 2 September 2015

Lab Notebook Overview

Week 1: May 2-8

Secretion

E.coli

NADPH Cofactor

Modeling

Comb

Week 2: May 9-15

Secretion

Expression

NADPH Cofactor

Modeling

Comb

Week 3: May 16-22

Secretion

Expression

NADPH Cofactor

Modeling

Comb

Week 4: May 23-29

Expression

NADPH Cofactor

Modeling

Comb

Week 5: May 30-June 5

Secretion

Expression

NADPH Cofactor

Modeling

Comb

Week 6: June 6-12

Secretion

Expression

NADPH Cofactor

Modeling

Comb

Week 7: June 13-19

Secretion

Expression

NADPH Cofactor

Modeling

Comb

Week 8: June 20-26

Expression

NADPH Cofactor

Modeling

Comb

Week 9: June 27-July 3

Secretion

Expression

NADPH Cofactor

Modeling

Comb

Week 10: July 4-10

Secretion

NADPH Cofactor

Modeling

Comb

Week 11: July 11-17

Expression

NADPH Cofactor

Modeling

Comb

Week 12: July 18-24

Secretion

Expression

NADPH Cofactor

Modeling

Comb

Week 13: July 25-31

Secretion

Expression

NADPH Cofactor

Modeling

Comb

Week 14: August 1-7

Secretion

Expression