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

Line 163: Line 163:
 
<div class="bacillus">
 
<div class="bacillus">
 
<h2><i>Bacillus</i></h2>
 
<h2><i>Bacillus</i></h2>
−
<p>We designed gblocks for our gene (3-α-HSD): one for the original sequence and one Bacillus-optimized.</p>
+
<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>
 
<p><i>Active team members this week: Liron, Tal, and Ruth</i></p>
 
</div>
 
</div>
Line 169: Line 169:
 
<h2><i>E.coli</i></h2>
 
<h2><i>E.coli</i></h2>
 
<p>Planned the Gibson reaction for our gBlock of 3α-HSD and the plasmid N155 (from our mentors) containing pT7, RBS and mcherry.</p>
 
<p>Planned the Gibson reaction for our gBlock of 3α-HSD and the plasmid N155 (from our mentors) containing pT7, RBS and mcherry.</p>
−
                     <p>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.</p>
+
                     <p>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.</p>
 
                     <p>Performed reverse PCR to the N155 plasmid and carried out a DPN1 reaction on the product.</p>
 
                     <p>Performed reverse PCR to the N155 plasmid and carried out a DPN1 reaction on the product.</p>
 
                     <p><i>Active team members this week: Adi and Alexey</i></p>
 
                     <p><i>Active team members this week: Adi and Alexey</i></p>
Line 180: Line 180:
 
<div class="comb">
 
<div class="comb">
 
<h2>Comb</h2>
 
<h2>Comb</h2>
−
<p>content</p>
+
<p>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.</p>
 +
<p><i>Active team members this week: Maayan</i></p>
 
</div>
 
</div>
 
<div class="nadph">
 
<div class="nadph">
 
<h2>NADPH</h2>
 
<h2>NADPH</h2>
−
<p>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.</p>
+
<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>
 
<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>
 
<p><i>Active team members this week: Yael and Roni</i></p>
 
<p><i>Active team members this week: Yael and Roni</i></p>
Line 196: Line 197:
 
<div class="bacillus">
 
<div class="bacillus">
 
<h2><i>Bacillus</i></h2>
 
<h2><i>Bacillus</i></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>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>
 
<p><i>Active team members this week: Liron, Tal, and Ruth</i></p>
 
</div>
 
</div>
Line 202: Line 203:
 
<h2>E.coli</h2>
 
<h2>E.coli</h2>
 
<p>Our gBlock arrived!!!</p>
 
<p>Our gBlock arrived!!!</p>
−
                     <p>We performed PCR on the 3αHSD gBlock with only 10 cycles (as recommended), but received no band in the gel</p>
+
                     <p>We performed PCR on the 3α-HSD gBlock with only 10 cycles (as recommended), but received no band in the gel.</p>
−
                     <p>We performed PCR on the 3αHSD gBlock in higher temperatures, but still received no bands in the gel</p>
+
                     <p>We performed PCR on the 3α-HSD gBlock in higher temperatures, but still received no bands in the gel.</p>
 
                     <p><i>Active team members this week: Adi</i></p>
 
                     <p><i>Active team members this week: Adi</i></p>
 
                                          
 
                                          
Line 213: Line 214:
 
<div class="comb">
 
<div class="comb">
 
<h2>Comb</h2>
 
<h2>Comb</h2>
−
<p>content</p>
+
<p>Met with Prof. Hovav Gazit and set a date for printing the comb.</p>
 +
<p><i>Active team members this week: Maayan</i></p>
 
</div>
 
</div>
 
<div class="nadph">
 
<div class="nadph">
Line 233: Line 235:
 
<div class="ecoli">
 
<div class="ecoli">
 
<h2>E.coli</h2>
 
<h2>E.coli</h2>
−
<p>Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.</p>
+
<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>
 
                     <p><i>Active team members this week: Alexey</i></p>
 
</div>
 
</div>
Line 242: Line 244:
 
<div class="comb">
 
<div class="comb">
 
<h2>Comb</h2>
 
<h2>Comb</h2>
−
<p>content</p>
+
<p>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.  </p>
 +
<p><i>Active team members this week: Maayan and Sagi</i></p>
 
</div>
 
</div>
 
<div class="nadph">
 
<div class="nadph">
 
<h2>NADPH</h2>
 
<h2>NADPH</h2>
−
<p>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.</p>
+
<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>
 
<p><i>Active team members this week: Yael and Roni</i></p>
 
</div>
 
</div>
Line 256: Line 259:
 
<div class="ecoli">
 
<div class="ecoli">
 
<h2>E.coli</h2>
 
<h2>E.coli</h2>
−
<p>Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.</p>
+
<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</i></p>
+
                    <p><i>Active team members this week: Alexey and Adi</i></p>
 
</div>
 
</div>
 
<div class="modeling">
 
<div class="modeling">
Line 265: Line 268:
 
<div class="comb">
 
<div class="comb">
 
<h2>Comb</h2>
 
<h2>Comb</h2>
−
<p>content</p>
+
<p>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.  </p>
 +
<p><i>Active team members this week: Maayan and Sagi</i></p>
 
</div>
 
</div>
 
<div class="nadph">
 
<div class="nadph">
Line 281: Line 285:
 
<div class="bacillus">
 
<div class="bacillus">
 
<h2>Bacillus</h2>
 
<h2>Bacillus</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 Bacillus genome).</p>
+
<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>
 
<p><i>Active team members this week: Liron, Adi, Yael, and Ruth</i></p>
 
</div>
 
</div>
 
<div class="ecoli">
 
<div class="ecoli">
 
<h2>E.coli</h2>
 
<h2>E.coli</h2>
−
<p>Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.</p>
+
<p>We ordered new primers for the 3αHSD gBlock.</p>
−
                     <p><i>Active team members this week: Alexey</i></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>
 +
                     <p><i>Active team members this week: Alexey and Adi</i></p>
 
</div>
 
</div>
 
<div class="modeling">
 
<div class="modeling">
Line 295: Line 300:
 
<div class="comb">
 
<div class="comb">
 
<h2>Comb</h2>
 
<h2>Comb</h2>
−
<p>content</p>
+
<p>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 .</p>
 +
<p><i>Active team members this week: Maayan and Sagi</i></p>
 
</div>
 
</div>
 
<div class="nadph">
 
<div class="nadph">
 
<h2>NADPH</h2>
 
<h2>NADPH</h2>
−
<p>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.</p>
+
<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>
 
<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>
 
<p><i>Active team members this week: Yael and Roni</i></p>
 
<p><i>Active team members this week: Yael and Roni</i></p>
Line 311: Line 317:
 
<div class="bacillus">
 
<div class="bacillus">
 
<h2>Bacillus</h2>
 
<h2>Bacillus</h2>
−
<p>We checked the plasmids we got from TAU and Weizmann Institute by sending for sequencing.</p>
+
<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>
 
<p><i>Active team members this week: Liron, Tal, and Ruth</i></p>
 
</div>
 
</div>
 
<div class="ecoli">
 
<div class="ecoli">
 
<h2>E.coli</h2>
 
<h2>E.coli</h2>
−
<p>Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.</p>
+
<p>We performed a PCR reaction again with the new primers.  We saw no bands in the gel.</p>
−
                                        <p><i>Active team members this week: Alexey</i></p>
+
<p>We planned a new strategy for cloning using restriction enzymes, with BBa_K784023 in pSB1C3 (from iGEM12_Technion) as the backbone.</p>
−
 
+
                    <p><i>Active team members this week: Alexey and Adi</i></p>
 
</div>
 
</div>
 
<div class="modeling">
 
<div class="modeling">
Line 326: Line 332:
 
<div class="comb">
 
<div class="comb">
 
<h2>Comb</h2>
 
<h2>Comb</h2>
−
<p>content</p>
+
<p>We printed the second prototype of the comb.  The flow that came out of the teeth of the comb was not simultaneous.</p>
 +
<p><i>Active team members this week: Maayan and Sagi</i></p>
 
</div>
 
</div>
 
<div class="nadph">
 
<div class="nadph">
 
<h2>NADPH</h2>
 
<h2>NADPH</h2>
−
<p>content</p>
+
<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>
 +
<p>Colony PCR to the colonies showed three positive colonies.  We sent one for sequencing.</p>
 +
<p><i>Active team members this week: Yael and Roni</i></p>
 
</div>
 
</div>
 
</div>
 
</div>
Line 340: Line 350:
 
<div class="bacillus">
 
<div class="bacillus">
 
<h2>Bacillus</h2>
 
<h2>Bacillus</h2>
−
<p>content</p>
+
<p>We designed primers for the 3a-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>
 
<div class="ecoli">
 
<div class="ecoli">
 
<h2>E.coli</h2>
 
<h2>E.coli</h2>
−
<p>Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.</p>
+
<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
−
                                        <p><i>Active team members this week: Alexey</i></p>
+
concentration, we cut the plasmid with SpeI and XbaI and performed a CIP reaction.</p>
−
 
+
<p>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.</p>
 +
                    <p><i>Active team members this week: Adi</i></p>
 
</div>
 
</div>
 
<div class="modeling">
 
<div class="modeling">
Line 354: Line 366:
 
<div class="comb">
 
<div class="comb">
 
<h2>Comb</h2>
 
<h2>Comb</h2>
−
<p>content</p>
+
<p>We searched for a microfluidics flow specialist and set up a meeting with Prof. Moran Bercovici from mechanical engineering faculty at Technion.</p>
 +
<p><i>Active team members this week: Maayan and Sagi</i></p>
 
</div>
 
</div>
 
<div class="nadph">
 
<div class="nadph">
 
<h2>NADPH</h2>
 
<h2>NADPH</h2>
−
<p>content</p>
+
<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>.
 +
<p><i>Active team members this week: Yael and Roni</i></p>
 
</div>
 
</div>
 
</div>
 
</div>
Line 366: Line 381:
 
<h1>Week 8: June 20-26</h1>
 
<h1>Week 8: June 20-26</h1>
 
<div class="week_content">
 
<div class="week_content">
−
<div class="bacillus">
 
−
<h2>Bacillus</h2>
 
−
<p>content</p>
 
−
</div>
 
 
<div class="ecoli">
 
<div class="ecoli">
 
<h2>E.coli</h2>
 
<h2>E.coli</h2>
−
<p>Performed PCR on the 3αHSD gBlock in lower temperatures, no bands in the gel.</p>
+
<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: Alexey</i></p>
+
                    <p><i>Active team members this week: Adi</i></p>
−
 
+
 
</div>
 
</div>
 
<div class="modeling">
 
<div class="modeling">
Line 382: Line 392:
 
<div class="comb">
 
<div class="comb">
 
<h2>Comb</h2>
 
<h2>Comb</h2>
−
<p>content</p>
+
<p>Meeting with Prof. Moran Bercovici, a specialist in microfluidic flow.  He gave us a lot of information about the flow inside the channels.</p>
 +
<p><i>Active team members this week: Maayan and Sagi</i></p>
 
</div>
 
</div>
 
<div class="nadph">
 
<div class="nadph">
 
<h2>NADPH</h2>
 
<h2>NADPH</h2>
−
<p>content</p>
+
<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>
 +
<p><i>Active team members this week: Yael and Roni</i></p>
 
</div>
 
</div>
 
</div>
 
</div>

Revision as of 12:12, 20 August 2015

Team: Technion 2015

Home
Navigate

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

Modeling

content

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

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

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

Modeling

content

Comb

Met with Prof. Hovav Gazit and set a date for printing the comb.

Active team members this week: Maayan

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

Active team members this week: Alexey

Modeling

content

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

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 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

Modeling

content

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

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

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

Modeling

content

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

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

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

Modeling

content

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

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

Week 7: June 13-19

Bacillus

We designed primers for the 3a-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

Modeling

content

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

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

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

Modeling

content

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

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

Week 9: June 27-July 3

Bacillus

content

E.coli

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

Active team members this week: Alexey

Modeling

content

Comb

content

NADPH

content

Week 10: July 4-10

Bacillus

content

E.coli

We took a break for our exams.

Modeling

content

Comb

content

NADPH

content

Week 11: July 11-17

Bacillus

content

E.coli

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

Active team members this week: Alexey

Modeling

content

Comb

content

NADPH

content

Week 12: July 18-24

Bacillus

content

E.coli

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

Active team members this week: Alexey

Modeling

content

Comb

content

NADPH

content

Week 13: July 25-31

Bacillus

content

E.coli

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

Active team members this week: Alexey

Modeling

content

Comb

content

NADPH

content

Week 14: August 1-7

Bacillus

content

E.coli

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

Active team members this week: Alexey

Modeling

content

Comb

content

NADPH

content

Week 15: August 8-14

Bacillus

content

E.coli

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

Active team members this week: Alexey

Modeling

content

Comb

content

NADPH

content

Week 16: August 15-21

Bacillus

content

E.coli

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

Active team members this week: Alexey

Modeling

content

Comb

content

NADPH

content

Week 17: August 22-28

Bacillus

content

E.coli

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

Active team members this week: Alexey

Modeling

content

Comb

content

NADPH

content

Week 18: August 29-September 4

Bacillus

content

E.coli

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

Active team members this week: Alexey

Modeling

content

Comb

content

NADPH

content

Week 19: September 5-11

Bacillus

content

E.coli

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

Active team members this week: Alexey

Modeling

content

Comb

content

NADPH

content

Week 20: September 12-18

Bacillus

content

E.coli

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

Active team members this week: Alexey

Modeling

content

Comb

content

NADPH

content

Contact Us