Team:British Columbia/Notebook/Week 7

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==July 18 2011==
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<html>
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'''Track: Beta-pinene synthase'''
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#bod {width:935px; float:left; background-color: white; margin-left: 15px;  margin-top:10px;}</style>
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<div id="bod"><b>Week 7: July 17-23</b> <html><a name="w7"></a></html>
 +
==Lab Meeting - July 18==
 +
Still figuring out accommodation and flight options..
 +
The team needs to continue fundraising!
-
Marianne's sequencing failed for all of P2SDM1, P2SDM2, P2SDM3. Marianne, very frustrated at this point, decided that she doesn't want to waste any more time on sequencing. So she restriction digested the miniprepped plasmids to see if the plasmids get cut (if the site is still there, then the vector should cut and give a single band).Gel verification of the digests suggested that the site has been removed (show bands that are identical to the uncut template/original vector).
+
Repeated sequencing at NAPS has been failing. The grad advisors (Alina and Rafael) are helping to troubleshoot. Is it NAPS? Are our samples contaminated? Rafael usually sends his samples to a sequencing company in Alabama; he suggests we send a few of our samples there first as they are supposed to be quite reliable.
-
==July 19 2011==
+
==Human Practices==
-
<b>1,8-cineole</b>
+
<html><a title='By Iwona Erskine-Kellie (Flickr) [CC-BY-2.0 (www.creativecommons.org/licenses/by/2.0)], via Wikimedia Commons' href='http://commons.wikimedia.org/wiki/File:Science_World_Sunset.jpg'><img height='200' alt='Science World Sunset' src='http://upload.wikimedia.org/wikipedia/commons/thumb/a/a0/Science_World_Sunset.jpg/800px-Science_World_Sunset.jpg'/></a></html>
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*Despite not getting any sequencing results, the restriction digest gels clearly showed that there were no cut sites in the synthase, so Jacob decided to try getting the synthases on biobricks. Today, Jacob ran a biobrick PCR to add the proper prefix and suffix to his synthase, but it did not work.  
+
-
<b>Miscellanea</b>
+
Laura was very excited to meet with Dr. Anderson on Monday to discuss our teams involvement in the Future Science Leaders program at Science World! Science World British Columbia is a non-profit organization which engages British Columbians in science and inspires future science and technology leadership throughout our province. The Future Science Leaders is a program offered to high school students who are keen and bright and want to further their understanding of science. Through weekly meetings the program will cover several difference sciences such as biology, mathematics, technology, physical sciences, earth and space, and technology. We as an iGEM team have the privilege of presenting our project to this group of high school students and their parents. We will also be educating the students on the diverse applications of synthetic biology and give a brief tutorial about what synthetic biology is! There is also an opportunity to promote the idea of a high school iGEM team for next year.
-
*New ampicillin plates were made, and the pag 413 GPD plasmid (for putting our synthases in yeast) was amplified. After the plasmid was amplified, the team decided to use another plasmid, making the amplified pag 413 GPD plasmid irrelevant for the project.
+
-
<b>ERG20</b>
+
==3-Carene==
-
Gurpal was working with the erg20 gene and trying to synthesize the erg20-2 mutant from it on July 19th. After ordering and receiving primers (forward and backward), his goal today was to isolate the erg20 gene from wildtype yeast. Three tubes were prepared for the PCR, each containing a different amount of yeast genome, and the PRC was initiated at 2:20pm. When the PRC was finished, the tubes were incubated in a 37 degree incubator for 1.5 hours. Afterwards, Jacob ran a gel electrophoresis experiment to determine if the site was cleaved. However, the gel showed that nothing except our ladder. Therefore, this experiment failed.
+
[[File:Marijacob1.JPG | thumb | right | 200px | Jacob working away]]
 +
Daisy has been trying to put the his-tagged truncated 3-carene synthase into the yeast plasmid, PA415GPD. This has been more difficult than anticipated. The primers used to PCR out the truncated 3-carene synthase seem to produce a smear around 1.7 kb. This is the correct size range for the truncated synthase but it is not a clear band. Daisy may need to adjust the annealing temperature of her primers to get a specific band. At this point, she is going to digest and ligate this non-specific band into the yeast plasmid. She will try purification methods of gel extraction and PCR purification...
-
Fortunately, the erg20 and erg20-2 genes arrived from France on pNEV-N plasmids on July 20th! This greatly increased the entire team's morale 10 fold!
 
-
On July 21st, Gurpal transformed the erg20 and erg20-2 genes into e-coli to make more copies. The erg20 gene was on a pBS plasmid and the erg20-2 was on the pKS plasmid. He followed the protocol in the lab and made 4 plates:
+
==Characterization: Limonene Synthase==
-
plate 1: pBS plasmid + ampicillin + LB agar
+
Daisy has decided to characterize the limonene synthase and the IDI1 (with Jacob) to fulfill the gold medal criteria. A couple of important points:
-
plate 2: pKS plasmid + ampicillin + LB agar
+
-
plate 3: ampicillin + LB agar
+
-
plate 4: LB agar
+
-
The plates were grown overnight. On July 22nd, the first plate and second plates were observed to have ln (too many colonies) and the controls were empty - as expected. However, this experiment was not sufficient because there was no positive control.  
+
-
Gurpal repeated the transformation protocol and used a control with ampicillin resistance. This time, he made brand new ampicillin LB agar plates and plated 4 of them. The plates contained:
+
-Limonene synthase is from '''lemon'''
-
plate 1: pKS plasmid + ampicillin + LB agar
+
-
plate 2: pBS plasmid + ampicillin + LB agar
+
-
plate 3: pg plasmid (we already know it grows in ampicillin) + ampicillin + LB agar
+
-
plate 4: competent cells + LB agar
+
-
We expected plates 1-3 to grow single colonies and plate 4 to contain nothing. Gurpal grew the plates overnight for 18 hours. On July 23rd, the experiment was proven to be a success because plates 1-3 contained single coloneis and plate 4 contained nothing.
+
-
In the afternoon of July 23rd, Gurpal prepared 5 overnight cultures.  
+
-Limonene synthase is a composite part (ie. Has other genes that supposedly increase terpenoid biosynthesis)
-
tube 1: pKS plasmid + LB broth + ampicillin
+
-
tube 2: pKS plasmid + LB broth + ampicillin
+
-Limonene synthase is on an ampicillin resistant plasmid
-
tube 3: pBS plasmid + LB broth + ampicillin
+
-
tube 4: LB broth (control) --> he expected it to grow nothing
+
-
tube 5: LB broth + ampicillin (control) --> he expected it to grow nothing
+
-
On July 24th, the experiment was proven to be a success because the control tubes (4 and 5) were clear liquids. This meant that nothing grew in them.
+
-
In the afternoon of July 24th, Gurpal mini prepped his overnight cultures using a PureLink Quick Plasmid Miniprep Kit and isolated pBS and pKS DNA plasmids. In particular, the concentration was determined using a nanodrop:
+
-We are not sure how well the synthase will grow in bacteria because the synthase comes from plants
-
pBS: 100.5 microg/microL
+
-
pKS: 90.0 microg/microL
+
-
pKS: 69.2 microg/microL
+
-
In order to verify the transformation was a success, Gurpal and Jacob performed a gel electrophoresis experiment. The gel columns were:
+
-
column 1: purified pBS plasmid
+
-
column 3: purified pKS plasmid
+
-
column 5: purified pKS plasmid
+
-
column 7: 1kb DNA ladder
+
-
The results were columns 1, 3 and 5 each showed up around 7.5 kb. This proves the transformation is a success because both the pKS and pBS plasmids were approximately 7 kb in size.
+
 +
-We plan to incorporate '''pRARE2''' plasmid into the bacteria also. The pRARE2 has rare codons that allow the bacteria to express synthases. This is from Bohlmann Lab (Chris Keeling - Thanks!)
 +
-We plan to add the '''IDI1''' part from the registry into the bacteria also
-
'''3-Carene'''
+
-We plan to improve the '''IDI1''' part by removing a cut site (done by Jacob)
-
Daisy has been trying to put the his tagged truncated 3-carene synthase into the yeast plasmid, PA415GPD. This has been more difficult than anticipated. The primers used to PCR out the truncated 3-carene synthase seem to produce a smear-like band at around 1.7 kb. This is the correct size range for the truncated synthase but it is not a clear definite line. Daisy may need to adjust the annealing temperature of her primers to try and get a specific band. At this point, she is going to try and digest and ligate this non specific band into the yeast plasmid. She will try purification methods of gel extraction and PCR purification to try and make the ligation work.
+
-IDI1 gene supposedly increases terpenoid biosynthesis
-
Daisy is also starting on the characterization of a limonene synthase in bacterial expression systems.
+
==1,8-Cineole==
-
==July 20  2011==
+
Jacob's sequencing also came back null. However, the restriction digest gels clearly showed that there were no cut sites in the synthase, so Jacob decided to try getting the synthases into biobrick plasmids. Three PCR attempts later...
-
'''Track: Beta-pinene synthase'''
+
-
Marianne PCR'ed off the original and the SDM'ed synthase using yeast primers (contains XhoI and SpeI restriction enzyme sites) in order to put it in the yeast plasmid. Marianne also PCR'ed off the SDM'ed synthase using biobrick primers (contains EcoRI, XbaI, SpeI, PstI restriction enzyme sites) in order to put it in the psb1C3 backbone. Gel verification for both PCRs shows bands at the correct length!
+
==beta-Pinene==
-
<b>(-)-limonene</b>
+
[[File:Marijacob2.JPG | thumb | left | 150px |Marianne and Jacob share their frustrations]]
-
*PCR off the synthase using yeast primers (contains SpeI and NotI restriction enzyme sites) in order to put into the yeast plasmid
+
-
*Gel veriification shows 4 bands - PCR failed
+
-
*SDM PCR the synthase using new primers
+
Marianne's sequencing attempts failed. Very frustrated at this point, she decided that she doesn't want to waste any more time on sequencing. So she restriction digested the mini-prepped plasmids to see if the site is still there. Gel verification of the digests suggested that the site has been removed (the gel showed bands identical to the uncut plasmid).
-
==July 21 2011==
+
Marianne PCRed off the original and the SDMed synthase using yeast primers (containing XhoI and SpeI restriction enzyme sites) in order to put it in the yeast plasmid PA415GPD. Marianne also PCRed off the SDMed synthase using biobrick primers (containing EcoRI, XbaI, SpeI, PstI restriction enzyme sites) to put it in the psb1C3 backbone. Gel verification for both PCRs shows bands at the correct length!
-
<b>(-)-limonene</b>
+
Marianne started to assemble the biobrick part. She digested the linearized psb1C3 and the synthase gene with EcoRI and PstI using Biobrick Restriction Digest protocol. She then ligated the backbone and the part and transformed it into DH5alpha competent cells. Colony PCR of transformants using G1004 and G1005 primers resulted in no bands on the gel... Therefore, she repeated the PCR using a new batch of primers as well as a different primer pair VF2/VR.  
-
*DpnI digest yesterday's SDM PCR products at 37C for 4 hours
+
<br><br>
-
*Gel verification of SDM PCR products show no bands. PCR failed.
+
-
*PCR off the synthase to put onto yeast plasmid
+
Marianne also restriction digested the yeast plasmids and synthase genes and ligated the following 8 combinations:
-
**Troubleshooting: Use gradient, from 64C to 72C
+
*original + his-tagged + GAL
-
*Gel verification of PCR products for all samples show 4 bands (same as yesterday). This may indicate, again, unspecific binding.
+
*original + his-tagged + GDP
 +
*original + no-his + GAL
 +
*original + no-his + GDP
 +
*SDM + his-tagged + GAL
 +
*SDM + his-tagged + GDP
 +
*SDM + no-his + GAL
 +
*SDM + no-his + GDP
 +
Marianne then transformed the ligated samples. Marianne had a very long day...
-
'''Track: Beta-pinene synthase'''
+
==(-)-Limonene==
-
Marianne started to assemble the biobrick part. She digested the linearized psb1C3 and the synthase (part) with EcoRI and PstI using Biobrick Restriction Digest protocol. She then ligated the backbone and the part using Biobrick Ligation protocol. Finally, the ligation was transformed into DH5 alpha competent cells.
+
While the SDMs were failing, Vicki decided to try to be efficient by running another experiment in parallel: She decided to use the yeast primers to get the original (un-SDMed) synthase out, and into a yeast plasmid. However, PCR of the synthase using yeast primers resulted in 4 bands on all the lanes the gel (unexpected results! Vicki should expect a band at around 2kb).  
 +
*Troubleshooting: use a temperature gradient from 64oC to 72oC... Result: 4 bands again.
 +
*More troubleshooting: decrease the extension time to 30sec (15sec/1kb) with an annealing temperature of 70C. Vicki will also transform original plasmid (pADM743) into DH5alpha cells in case the plasmid she is working with has been contaminated somehow, and hence not being PCRed off properly.
-
<b>1,8-cineole</b>
+
On the other note, Vicki decided to get new primers (higher temperature and more base pairs to anneal to the template) to re-attempt the SDM... but once again, there were no bands. Vicki is extremely frustrated at this point, and is starting to doubt the integrity of the original tube (pADM743) of (-)-limonene synthase.
-
*Assembling biobrick part.  
+
-
**Second PCR did not work
+
-
***Third PCR set to run overnight
+
-
==July 22 2011==
+
==ERG20 & erg20-2==
-
<b>Human Practices</b>
+
Since they still hadn't received the plasmids from France, Gurpal decided to try generating the erg20-2 mutant by SDM. After ordering and receiving primers, his goal was to isolate the ERG20 gene from wild type yeast. Three tubes were prepared for the PCR, each containing a different amount of yeast genome, and the PCR was performed. Afterwards, Jacob ran gel electrophoresis to see if the PCR worked. However, the gel showed nothing but the ladder... just for kicks, here the is the proof:
-
Laura is very excited to meet with Mrs. Anderson on Monday to discuss our teams involvement in the Future Science Leaders program at Science World! Science World British Columbia is a non-profit organization which engages British Columbians in science and inspires future science and technology leadership throughout our province. The Future Science Leaders is a program offered to high school students who are keen and bright and want to further their understanding of science. Through weekly meetings the program will cover several difference sciences such as biology, mathematics, technology, physical sciences, earth and space, and technology. We as an iGEM team have the privilege of presenting our project to this group of high school students and their parents. We will also be educating the students on the diverse applications of synthetic biology and give a brief tutorial about what synthetic biology is! There is also an opportunity to promote the idea of a high school iGEM team for next year. I am anticipating this meeting and hope to get some feedback and advice for other related Human Practice projects.
+
-
<b>(-)-limonene</b>
+
[[File: Gel_-_attempt_to_remove_ERG20.jpg‎| frame | center | ]]
-
*Re-do PCR to get the synthase off of the pET101 plasmid onto the yeast plasmid
+
-
**Troubleshooting: Decrease the extension time to 30sec (15sec/1kb) with an annealing temperature of 70C
+
-
*Transform original plasmid (pADM743) into DH5alpha cells
+
Fortunately, the ERG20 and erg20-2 genes arrived from France on pNEV-N plasmids the next day! This increased the entire team's morale 10-fold! The ERG20 gene was on the pBS plasmid and the erg20-2 was on the pKS plasmid. Gurpal successfully transformed these plasmids into <i>E. coli</i> on his second attempt using newly made Ampicillin agar plates.
 +
In order to replicate more cells, Gurpal made overnight cultures for both the pBS and pKS plasmids in E. coli. The following day, I had performed mini plasmid preps (using the Invitrogen kit) and had one sample of pBS plasmid and 2 samples of pKS plasmids. He used the nanodrop and determined that the concentrations were as follows:
 +
[pBS] = 100.5 ng/µL
 +
[pKS] = 90.0 ng/µL (first sample, labeled pKS1)
 +
[pKS] = 69.2 ng/µL (second sample, labeled pKS2)
-
'''Track: Beta-pinene synthase'''
+
Gurpal verified that these were in fact the correct genes. Each gene should have been about 7000 bps. After performing gel electrophoresis, he verified that his transformations and mini plasmid preps were a success!
 +
Here is the gel image:
-
Marianne conducted colony PCR on the colonies from biobrick plates (using G1004,G1005 primers), but gel verification showed no bands. Therefore, she cPCR the same colonies from biobrick plates using a new batch of G1004/G1005 primers as well as VF2/VR.  
+
[[File:Gel_-_verification_of_pBS_pKS_in_ecoli.jpg‎‎ | frame | center | ]]
-
Marianne also restriction digested the yeast plasmids and synthase and ligated the following 8 versions together:
+
==alpha-Pinene==
-
*original + his-tagged + GAL
+
-
*original + his-tagged + GDP
+
-
*original + no-his + GAL
+
-
*original + no-his + GDP
+
-
*SDM + his-tagged + GAL
+
-
*SDM + his-tagged + GDP
+
-
*SDM + no-his + GAL
+
-
*SDM + no-his + GDP
+
-
Marianne then transformed the ligated samples. Marianne had a very long day...
+
Joe is taking a break from lab work because he has a poster day presentation to prepare for at his other summer research lab at the Child and Family Research Institute (CFRI). In the mean time, he has started looking at the arcGIS data from the B.C forestry on the pine-beetle spread while learning how to use the arcGIS software. He is also waiting for primers to come so that he can introduce cut-sites to his alpha-pinene synthase gene for PCR, in preparation for insertion into our chosen yeast constructs <html><b><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K517000"> GAL promoter BBa_k517000 </b></a></html> and <html><b><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K517001"> GPD promoter BBa_k517001 </b></a></html>
 +
 
 +
 
 +
<html>
 +
<a href="https://2011.igem.org/Team:British_Columbia/Notebook"><center><b>Back to the Notebook</b></center></a>

Latest revision as of 23:12, 16 October 2011

Team: British Columbia - 2011.igem.org

Week 7: July 17-23

Contents

Lab Meeting - July 18

Still figuring out accommodation and flight options.. The team needs to continue fundraising!

Repeated sequencing at NAPS has been failing. The grad advisors (Alina and Rafael) are helping to troubleshoot. Is it NAPS? Are our samples contaminated? Rafael usually sends his samples to a sequencing company in Alabama; he suggests we send a few of our samples there first as they are supposed to be quite reliable.

Human Practices

Science World Sunset

Laura was very excited to meet with Dr. Anderson on Monday to discuss our teams involvement in the Future Science Leaders program at Science World! Science World British Columbia is a non-profit organization which engages British Columbians in science and inspires future science and technology leadership throughout our province. The Future Science Leaders is a program offered to high school students who are keen and bright and want to further their understanding of science. Through weekly meetings the program will cover several difference sciences such as biology, mathematics, technology, physical sciences, earth and space, and technology. We as an iGEM team have the privilege of presenting our project to this group of high school students and their parents. We will also be educating the students on the diverse applications of synthetic biology and give a brief tutorial about what synthetic biology is! There is also an opportunity to promote the idea of a high school iGEM team for next year.

3-Carene

Jacob working away

Daisy has been trying to put the his-tagged truncated 3-carene synthase into the yeast plasmid, PA415GPD. This has been more difficult than anticipated. The primers used to PCR out the truncated 3-carene synthase seem to produce a smear around 1.7 kb. This is the correct size range for the truncated synthase but it is not a clear band. Daisy may need to adjust the annealing temperature of her primers to get a specific band. At this point, she is going to digest and ligate this non-specific band into the yeast plasmid. She will try purification methods of gel extraction and PCR purification...


Characterization: Limonene Synthase

Daisy has decided to characterize the limonene synthase and the IDI1 (with Jacob) to fulfill the gold medal criteria. A couple of important points:

-Limonene synthase is from lemon

-Limonene synthase is a composite part (ie. Has other genes that supposedly increase terpenoid biosynthesis)

-Limonene synthase is on an ampicillin resistant plasmid

-We are not sure how well the synthase will grow in bacteria because the synthase comes from plants

-We plan to incorporate pRARE2 plasmid into the bacteria also. The pRARE2 has rare codons that allow the bacteria to express synthases. This is from Bohlmann Lab (Chris Keeling - Thanks!)

-We plan to add the IDI1 part from the registry into the bacteria also

-We plan to improve the IDI1 part by removing a cut site (done by Jacob)

-IDI1 gene supposedly increases terpenoid biosynthesis

1,8-Cineole

Jacob's sequencing also came back null. However, the restriction digest gels clearly showed that there were no cut sites in the synthase, so Jacob decided to try getting the synthases into biobrick plasmids. Three PCR attempts later...

beta-Pinene

Marianne and Jacob share their frustrations

Marianne's sequencing attempts failed. Very frustrated at this point, she decided that she doesn't want to waste any more time on sequencing. So she restriction digested the mini-prepped plasmids to see if the site is still there. Gel verification of the digests suggested that the site has been removed (the gel showed bands identical to the uncut plasmid).

Marianne PCRed off the original and the SDMed synthase using yeast primers (containing XhoI and SpeI restriction enzyme sites) in order to put it in the yeast plasmid PA415GPD. Marianne also PCRed off the SDMed synthase using biobrick primers (containing EcoRI, XbaI, SpeI, PstI restriction enzyme sites) to put it in the psb1C3 backbone. Gel verification for both PCRs shows bands at the correct length!

Marianne started to assemble the biobrick part. She digested the linearized psb1C3 and the synthase gene with EcoRI and PstI using Biobrick Restriction Digest protocol. She then ligated the backbone and the part and transformed it into DH5alpha competent cells. Colony PCR of transformants using G1004 and G1005 primers resulted in no bands on the gel... Therefore, she repeated the PCR using a new batch of primers as well as a different primer pair VF2/VR.

Marianne also restriction digested the yeast plasmids and synthase genes and ligated the following 8 combinations:

  • original + his-tagged + GAL
  • original + his-tagged + GDP
  • original + no-his + GAL
  • original + no-his + GDP
  • SDM + his-tagged + GAL
  • SDM + his-tagged + GDP
  • SDM + no-his + GAL
  • SDM + no-his + GDP

Marianne then transformed the ligated samples. Marianne had a very long day...

(-)-Limonene

While the SDMs were failing, Vicki decided to try to be efficient by running another experiment in parallel: She decided to use the yeast primers to get the original (un-SDMed) synthase out, and into a yeast plasmid. However, PCR of the synthase using yeast primers resulted in 4 bands on all the lanes the gel (unexpected results! Vicki should expect a band at around 2kb).

  • Troubleshooting: use a temperature gradient from 64oC to 72oC... Result: 4 bands again.
  • More troubleshooting: decrease the extension time to 30sec (15sec/1kb) with an annealing temperature of 70C. Vicki will also transform original plasmid (pADM743) into DH5alpha cells in case the plasmid she is working with has been contaminated somehow, and hence not being PCRed off properly.

On the other note, Vicki decided to get new primers (higher temperature and more base pairs to anneal to the template) to re-attempt the SDM... but once again, there were no bands. Vicki is extremely frustrated at this point, and is starting to doubt the integrity of the original tube (pADM743) of (-)-limonene synthase.

ERG20 & erg20-2

Since they still hadn't received the plasmids from France, Gurpal decided to try generating the erg20-2 mutant by SDM. After ordering and receiving primers, his goal was to isolate the ERG20 gene from wild type yeast. Three tubes were prepared for the PCR, each containing a different amount of yeast genome, and the PCR was performed. Afterwards, Jacob ran gel electrophoresis to see if the PCR worked. However, the gel showed nothing but the ladder... just for kicks, here the is the proof:

Gel - attempt to remove ERG20.jpg

Fortunately, the ERG20 and erg20-2 genes arrived from France on pNEV-N plasmids the next day! This increased the entire team's morale 10-fold! The ERG20 gene was on the pBS plasmid and the erg20-2 was on the pKS plasmid. Gurpal successfully transformed these plasmids into E. coli on his second attempt using newly made Ampicillin agar plates.

In order to replicate more cells, Gurpal made overnight cultures for both the pBS and pKS plasmids in E. coli. The following day, I had performed mini plasmid preps (using the Invitrogen kit) and had one sample of pBS plasmid and 2 samples of pKS plasmids. He used the nanodrop and determined that the concentrations were as follows: [pBS] = 100.5 ng/µL [pKS] = 90.0 ng/µL (first sample, labeled pKS1) [pKS] = 69.2 ng/µL (second sample, labeled pKS2)

Gurpal verified that these were in fact the correct genes. Each gene should have been about 7000 bps. After performing gel electrophoresis, he verified that his transformations and mini plasmid preps were a success! Here is the gel image:

Gel - verification of pBS pKS in ecoli.jpg

alpha-Pinene

Joe is taking a break from lab work because he has a poster day presentation to prepare for at his other summer research lab at the Child and Family Research Institute (CFRI). In the mean time, he has started looking at the arcGIS data from the B.C forestry on the pine-beetle spread while learning how to use the arcGIS software. He is also waiting for primers to come so that he can introduce cut-sites to his alpha-pinene synthase gene for PCR, in preparation for insertion into our chosen yeast constructs GAL promoter BBa_k517000 and GPD promoter BBa_k517001


Back to the Notebook

Retrieved from "http://2011.igem.org/Team:British_Columbia/Notebook/Week_7"