Team:Lethbridge/Notebook/Lab Work/Justin

From 2011.igem.org

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<image src="https://static.igem.org/mediawiki/2011/1/14/Justina.jpg" height="400px"/>
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</p>
</p>
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=Week 3 (16-22)=
+
=Week 3 (May 16-22)=
-
==Manday==
+
==Monday==
<p>Check to see if Group 1 has successfully assembled biobricks by running samples on a 1% agarose gel followed by analyzing.  
<p>Check to see if Group 1 has successfully assembled biobricks by running samples on a 1% agarose gel followed by analyzing.  
</p>
</p>
Line 141: Line 135:
==Sunday==
==Sunday==
-
<p>Express BamH1 restriction endonuclease in E. coli DH5α in comparison to wild type E. coli DH5α that were not induced. BamH1 restriction endonuclease was induced with arabinose. Optical density measurements were taken and a growth curve of both cultures was generated.  
+
<p>Express BamH1 restriction endonuclease in E. coli DH5α in comparison to wild type E. coli DH5α that were not induced. BamH1 restriction endonuclease was induced with arabinose. Optical density measurements were taken and a growth curve of both cultures was generated. Also samples after induction were taken to run on an SDS-PAGE.
</p>
</p>
-
=Week 4 (23-29)=
+
=Week 4 (May 23-29)=
-
==Manday==
+
==Monday==
<p>
<p>
 +
Justin recuperated after working through the weekend.
</p>
</p>
==Tuesday==
==Tuesday==
-
<p>
+
<p>A 15% SDS-PAGE of samples taken from the BamH1 over-expression was ran. Gel looks promising ☺
</p>
</p>
==Wednesday==
==Wednesday==
<p>
<p>
 +
Parts that were constructed recently were PCR amplified.
</p>
</p>
==Thursday==
==Thursday==
 +
<p>An agarose gel was ran of the PCR products to determine which parts had the correct size. Parts that had an appropriate size range were sent for sequencing.
 +
Parts sent for sequencing:
 +
*1) ROO10-BOO34
 +
*2) K249002-BOO15
 +
*3) K331033-K331035
 +
 +
</p>
 +
 +
==Friday==
 +
<p>Justin assisted team #1
 +
</p>
 +
 +
==Saturday==
 +
<p> Time was spent researching, and assisting teams that spent the weekend working in lab.
 +
</p>
 +
 +
=Week 5 (May 30-31 and June 1-5)=
 +
 +
==Monday==
<p>
<p>
 +
Colones were picked from Morgan's (group 2) plates from 05/27/11 and set up for an overnight culture.
 +
</p>
 +
 +
==Tuesday==
 +
<p>
 +
Overnight cultures from May 30, 2011 were mini-preped using the QIAGEN spin column method. Then the DNA was PCR amplified and ran on a 1% agarose gel. Parts that were sent for sequencing were:
 +
*1) pBAD-P0440
 +
*2) K249002-B0015
 +
</p>
 +
<p> Assembly of 3 BioBricks!
 +
*1) pBad-rbs + lumazine-dt
 +
*2) K331033 + K331035
 +
*3) R0010 + B0034
 +
**--> We want to have pLacU-rbs-lumazine-dt, but since we have pbad and rbs we will use above. This will result in expressing lumazine synthase ASAP :)</p>
 +
<p> Dustin helped out with construction of parts</p>
 +
 +
==Wednesday==
 +
<p>Justin helped out team 3
 +
</p>
 +
 +
==Thursday==
 +
<p>Justin figured out what we need to order for lab, and compiled sequencing results.</p>
 +
<p> Set up a PCR of Assemblies from May 31 to run on an agarose gel of constructs.
</p>
</p>
==Friday==
==Friday==
 +
<p>Ran PCR products from June 2 on an agarose gel and retransformed DNA in DH5α
 +
</p>
 +
<p>Transform  DNA that will be sent for sequencing on Monday (suspected BioBricks). Parts that will be transformed:
 +
*1) K249002 + B0015
 +
*2) pBAD + P0040
 +
*3) R0010 + B0015
 +
*4) K249002 + B0015
 +
*5) K331033 + K331035
 +
</p>
 +
==Saturday==
 +
<p> All plates from transformations had some white colonies. Ranged from a lawn of white colonies to very few white colonies. White colonies were picked off each plate and a overnight culture was setup.</P>
 +
 +
==Sunday==
<p>
<p>
 +
Dee ran an agarose gel of several parts (Ryan pulled these out of glycerol stocks) to determine which parts should be sent for sequencing.
 +
</p>
 +
=Week 6 (June 6-12 )=
 +
 +
==Monday==
 +
<p> Cleaning the lab up a bit. So much work on our project has been done so far. Go team!
 +
 +
</p>
 +
 +
==Tuesday==
 +
<p> Helping Ryan figure out some PCR issues
 +
</p>
 +
 +
==Wednesday==
 +
<p> Reading synthetic biology papers and preparing for SynBio 5.0! Also getting some autoclaving done.
 +
</p>
 +
 +
 +
==Friday==
 +
<p>Attending Journal club at the University of Lethbridge. Got some great ideas
</p>
</p>
==Saturday==
==Saturday==
 +
<p> Mini-preped 5 samples/ colonies that Nathan assembled  (pBAD-rbs-lumazine-dT)  using QIAGEN mini-prep method. Then the parts were PCR amplified, and will later be restricted and ran on a agarose gel.</P>
 +
==Sunday==
 +
<p> PCR products from June 11, 2011 were ran on a 1.5% agarose gel to confirm their size</p>
 +
 +
=Week 7 (June 13- 19)=
 +
 +
<p>Justin left for ISMOS-3 on June 12, 2011. Had a great time on June 13,2011 listening to the keynote Steve Larter for the University of Calgary on "Microbes or Mass Transport -What are the Real Barriers to Production Scale Microbial Gasification of Oil or Coal to Produce Methane or Hydrogen in Subsurface Reservoirs?" The conference was very informative applied microbiology and molecular biology research that is occurring in the oil sands.</p>
 +
 +
<p>Then Justin left for SynBio 5.0 held at Stanford University. "I Attended SynBio 5.0, an international conference held this year at Stanford University in Palo Alto, California. At this conference, fellow team members and I got to see some of the leading experts in the field of synthetic biology and some of the amazingly creative research they are doing. Considering my love for iGEM, this conference gave me a whole new passion for this years iGEM competition." - Justin Vigar (transcribed by Harland Brandon)</p>
 +
 +
=Week 8 (June 20-26)=
 +
 +
==Monday==
<p>
<p>
 +
Planed out experimental protocol for over-expressing CFP with an agranine (10) tag (K331033) and purification using cation exchange
 +
</p>
</p>
 +
==Tuesday==
 +
<p>Gathered materials needed for over-expresion and purification of CFP with an arg tag. Made buffers for purification.
 +
</p>
 +
 +
==Wednesday==
 +
<p>
 +
Setup overnight culture of CFP with arg tag (K331033) for over-expression.
 +
</p>
 +
 +
==Thursday==
 +
<p>Over-expressed CFP with arg tag (K331033). Cell were frozen for purification down the road.
 +
</p>
 +
=Week 9 (June 26-30 )=
 +
 +
==Monday==
 +
<p> Calibrating pipettes, filling tip boxes and autoclaving. And mopping floor, Woo!
 +
 +
</p>
 +
 +
==Tuesday==
 +
<p> Aided team # 2
 +
</p>
 +
 +
==Wednesday==
 +
<p> PCR amplified
 +
*1) lumazine synthase-dt from glycerol stocks
 +
*2) R0010-B0034 assembly
 +
*3) R0010 in pSB1A2 (control)
 +
*4) K331035
 +
*5) K331033
 +
*6) pSB1A3 (J04450)
 +
*7) lumazine synthase
 +
</p>
 +
<p> Then Ran PCR products on a 2% agorose gel.</p>
 +
<p> constructs that were the correct size were:
 +
*1) Lumazine synthase
 +
*2) lumazine synthase-dt
 +
**the rest were undetermined.
 +
 +
==Thursday==
 +
Attempted purification of CFP arg (K331033) tagged protein. There are some issues that need to be solved.</p>
 +
 +
==Friday==
 +
<p>Problem solving and working on wiki.
 +
</p>
 +
=Week 10 (July 5-10)=
 +
 +
==Tuesday==
 +
<p>Clone J04500, pBAD-B0034, and K346007 into E. coli DH5α
 +
</p>
 +
==Wednesday==
 +
<p> Pick colonies of cloned parts, July 6, 2011, and started overnight culture
 +
</p>
 +
==Thursday==
 +
<p>Express BamH1 and show that growth is hindered; Minipreped cell cultures that were grown overnight to determine quality/quantity/size of DNA of certain parts; Assebled pLacI-RBS with lumazine-dT. Also a growth curved of a induced (arabinose) and un-induced culture were generated from optical density measurements.
 +
</p>
 +
<p> After 8 hours dilutions were made from the induced and un-induced cultures and plated.</p>
 +
<p><html><a href="https://2011.igem.org/Team:Lethbridge/Results#Results_7"><font color="blue">Results</font></a></html></p>
 +
 +
==Friday==
 +
<p> Mini-prep cell cultures that were grown overnight, by Nathan, to determine the quality/quaintly/ size of DNA of parts:
 +
*1) J04500
 +
*2) pBAD-B0034
 +
*3) K346007 </p>
 +
 +
<p>Mini-preped samples were restricted then ran on a 1% agarose gel. We were unable to detect DNA for pBAD-B0034 on the agarose gel. All other parts were visible and had an acceptable size.</p>
 +
==Saturday==
 +
<p> Continue assembly on our lumazine BioBrick, which I'm really excited about. I think we will be able to construct our whole massive construct with lumazine synthase and the fluorescent proteins. It will be so cool if we can show that the construct will express the proteins and work as a true interchangeable part!!!</p>
 +
<p> Assembly of pLacI-rbs (J04500) with lumazine.</p>
==Sunday==
==Sunday==
 +
<p> Transformation of yesterdays work! Transformed lumazine construct into  E. coli DH5α.</p>
 +
 +
=Week 12 (July 17-24)=
 +
 +
==Wednesday==
 +
<p>Determine if xyl transformations were successful and if K346007 was successfully transferred from pSB1C3 to pSB1K3. This was done by restricting the mini-preps and running a 1% agarose gel. It looks like we might have successfully assembled for the gel:
 +
*pSB1A3 promotor-rbs-xylJ-dt
 +
*pSB1A3 promoter-rbs-xylE-dt
 +
* pSB1A3 promoter-rbs-xylF
 +
*pSB1A3 promoter-rbs-xylk
 +
*pSB1A3 promoter-rbs-xylQ
 +
*pSB1A3 promoter-rbs-xylE-pSB1A3 promoter-rbs-xylQ
 +
*pSB1A3 promoter-rbs-xylE-pSB1A3 promoter-rbs-xylF
 +
*pSB1A3 promoter-rbs-xylE-pSB1A3 promoter-rbs-xylJ
 +
*pSB1A3 promoter-rbs-xylE-pSB1A3 promoter-rbs-xylK
 +
*J04450 pSB1C3
 +
*J04450 psb1k3
 +
*K346007 psB1K3
 +
These will need to be sequence to confirm they are the parts we believe them to be.
 +
</P>
 +
 +
==Thursday==
 +
<p>Today Team Justin determined if the assembly of R0010 (placI-rbs) and IC346007 (Agn43) was successful. Note: Boris did this assembly 07/20/11, see team 1 book for details. Plates that were transformed with parts do have white colonies :). These white colonies were selected and colony PCR was performed using the Prefix and Suffix primers.
 +
</p>
 +
 +
==Friday==
 +
<p> Samples from colony PCR (yesterday) were ran on a 1% agarose gel. Amplification was successful! However, no inserts of right size are present.   
 +
</P>
 +
 +
=Week 13 (July 25-31)=
 +
==Monday==
<p>
<p>
 +
Assemble K331035-K331033 and R0010-luma-dT</p>
 +
First what happened is the BioBricks were restricted out of the plasmids. Then to determine the concentration of DNA after the restrictions a 1% agarose gel was ran.
 +
 +
<p>The team has made so much progress, we are all very excited for Indianapolis! We had such a successful team meeting:) GO TEAM!
</p>
</p>
-
<br><br>
+
 
 +
==Tuesday==
 +
<p> Determine DNA concentration of J04500, K331033, K331035, and lumazine synthase dt. Team JV (Justin and Mr. Vigar) concluded that DNA concentration was too low for the spectrophotometer to read. However, the DNA was still assembled and transformed (by Ben).</p>
 +
<p> Then K331033 was ligated with K331035, and J04500 with lumazine-dt. Constructs were transformed into E. coli DH5α cells 
 +
</p>
 +
 
 +
==Wednesday==
 +
<p>Colonies were counted from yesterdays transformation. Both constructs resulted in white colonies --> We'll perform colony PCR on these samples see July 27, 2011
 +
</P>
 +
 
 +
 
 +
=Week 14 (August 1-7)=
 +
==Tuesday==
 +
First day in a three day experiment to isolate enhanced lumazine synthase from ''E. coli'' BL21(DE3) cells. Set up overnight culture, checked plasmids, made buffers.
 +
 
 +
==Wednesday==
 +
Second day, lumazine synthase is overexpressed. Open cells and run SDS-PAGE gel. Purification of lumazine synthase.
 +
 
 +
 
 +
==Saturday==
 +
Concentrated lumazine synthase. SDS-PAGE done to confirm this.
 +
 
 +
=Week 15 (August 8-14)=
 +
==Monday==
 +
Buffers made for chromatography. Two assemblies are ran for lumazine + inverter and inverter + tagged fluorescent proteins.
 +
 
 +
==Friday==
 +
Colony PCR of assemblies. Confirm inverter + tagged fluorescent.
 +
 
 +
=Week 16 (August 15-21)=
 +
==Tuesday==
 +
Picked colonies from plates to get more plasmids to assemble with.
 +
 
 +
=Week 17 (August 22-28)=
 +
==Tuesday==
 +
Assemblies of lumazine + inverter and fluorescent proteins.
 +
 
 +
==Wednesday==
 +
Transformation of yesterdays assemblies.
 +
 
 +
==Thursday==
 +
Helped out team 2 while Ryan is gone by testing assemblies of mms6 with polypetide signal tags and working with Dustin to assemble xylE with arginine polypeptide signal tag.
 +
 
 +
==Friday==
 +
Mms6 testes negative on an agarose gel.
 +
 
 +
=Week 18 (August 29 - September 4)=
 +
==Monday==
 +
Prepping cellular samples for electron micrograph imaging.
 +
 
 +
==Tuesday==
 +
Finished prepping samples for electron micrograph imaging.
 +
 
 +
==Friday==
 +
Images of cells expressing lumazine synthase taken.
 +
 
 +
 
 +
=Week 20 (September 12-18)=
 +
==Tuesday==
 +
LB cultures made from glycerol stocks.
 +
 
 +
==Wednesday==
 +
Mini-prepped cultures and assembled the isolated parts into psb1c3.
 +
 
 +
==Thursday==
 +
Continued assemblies from Wednesday.
 +
 
 +
==Friday==
 +
Continued assemblies from Wednesday and Thursday.
 +
 
 +
=Week 21 (September 19-25)=
 +
Prepared for aGEM in Edmonton
 +
 
 +
=Week 22 (September 26-28)=
 +
==Monday==
 +
Sent parts to the registry.
 +
 
 +
==Tuesday==
 +
Sent parts to the registry with Ryan.
 +
 
 +
==Wednesday==
 +
Gone to Calgary to give a presentation on the synthetic biology and the future of the oil sands.

Latest revision as of 03:50, 29 September 2011




Contents

Week 1 (May 2 - 8)

Monday

First day of offical work in the lab! I'm extremely excited for the next 4 months! Last night I inoculated numerous tubes containing 5 mL of LB media with cells from our glycerol stock in HJ's -80oC. The plasmid DNA was extracted from these cells using the QIAGEN spin column method. The purified plasmid DNA was analyzed and quantified using agarose gel electrophoresis. These DNA will be used for assemblies in the coming days.
I inoculated 4 2L flasks containing LB media with cells hosting biobrick plasmids. Tomorrow I plan to use a QIAGEN maxiprep protocol to obtain large amounts of these plasmids.
I finished the day organizing and cleaning the lab in preparation for this years volunteers to arrive. I've got two weeks to get the lab running, then they're here!

Tuesday

Our lab has received Maxiprep spin columns from BIOBASIC. Today I attempted to Maxiprep pSB1A3, pSB1C3, pSB1T3, and pSB1K3 for use this summer. After completing the protocol the samples were analyzed on an agarose gel, revealing little to no DNA. The columns won't be used again.

Wednesday

Harland inoculated 5 mL cultures containing E. coli hosting various biobricks last night. Today I minipreped the cells, digested the samples and ran them on an agarose gel to confirm the correct parts.

I spent the remainder of the day working on fundraising applications.

Thursday

Today I continued organizing the lab and autoclaved media in preparation for the 2011 iGEM volunteers to come. Before I left the lab I inoculated 5 mL LB media test tubes containing E. coli cells containg various biobricks that will be used to assemble our lumazine synthase / fluorescent protein construct.

Friday

I minipreped the E. coli cultures that were grown overnight. The parts were then assembled using the red/white screening protocol. Ligations were left overnight at room temperature.

Saturday

The ligations that were left overnight were transformed into E. coliDH5α cells. The transformed cells were plated on LB media pales with agar and incubated for 2 days

Week 2 (May 9 - 15)

Monday

Each plate from the transformations platted on Saturday showed a lawn of colonies. All colonies were white, none red. This is a highly unlikely result. A possible explanation could be that, the plates were improperly made and did not contain any antibiotic.

I divided a LB agar plate from the same batch as I used Saturday into 4 quadrants and streak plated E. coliDH5α cells containing different antibiotic resistant plasmids; pSB1A3, pSB1C3, pSB1T3, and pSC1K3. This should reveal which antibiotic is in the plates, if any

Tuesday

Everything grew on the plates, meaning no antibiotic was added to the plates. I threw out the faulty plates<p/> <p> The ligations from May 7th were re-transformed and plated on LB agar plates containing Tetracycline.

Wednesday

The assembly of the following constructs was attempted:

  • 1) pLacI-sRBS
  • 2) pBAD-sRBS
  • 3) K331033-K331035
  • 4) K331025-B0015
  • 5) R0040-K331029 into pSB1T3
Restrictions were done (standard iGEM restriction enzymes) and then the parts were ligated together.

Thursday

No cell growth was observed in the 5 mL cultures. Is there something wrong with our Tetracycline?

An Eckhart gel was done to screen the colonies on the plates from Tuesday. The resolution of the Eckhart gel was not optimal and it was hard to derive any meaningful information from this gel.

Constructs created from May 11, 2011 were transformed into E. coli DH5α cells. The transformations were plated on a new batch of tetracycline plates. Only two white colonies grew on one of the plates. Therefore, the DNA will be retransformed to determine if the quality of the DNA is good.

Friday

Constructs created on May 11, 2011 were retransformed into E. coli DH5α cells.

Saturday

Transformation from May 13, 2011 resulted in white colonies growing on tetracycline plates. These white colonies were mini-preped, so that an agarose gel could be ran to determine if we had successfully assembled our constructs.

Sunday

An agarose gel of mini-preped DNA was ran to determine if construction of constructs, May 11, 2011, was successful. DNA from mini-preps was restricted with EcoR1, and then as much of the post restriction mixture was loaded into the gel wells. Concluding from the gel: pSB1T3 was much larger than expected, and constructs that will be sent for sequencing are:

  • 1) pLacI-sRBS
  • 2) pBAD-sRBS
  • 3) K331025-B0015
  • 4) K331033-K331035

Week 3 (May 16-22)

Monday

Check to see if Group 1 has successfully assembled biobricks by running samples on a 1% agarose gel followed by analyzing.

Tuesday

More Plasmid Backbone and K331031 in pSB1C3 were made. Cells were incubated overnight at 37oC. Then the cells were mini-preped and ran on an ~1% agarose gel by Terrance. The gel was of poor quality and so they were re-ran.

Wednesday

Justin and Boris obtained the biobrick plasmid containing P0440. Then transformed this part into E. coli DH5α cells.

Thursday

Transformed cells were plated onto 2 plates. Plate #1 resulted in 5 colonies and Plate #2 4 colonies. A single colony was picked from each plate and incubated overnight at 37oC.

Friday

Boris and Justin mini-preped the overnight culture (May 19, 2011) of P0440 and Assemble the following constructs:

  • 1) pBAD-P0440
  • 2) R0010-B0034
  • 3) K331025-B0015
  • 4) K249002-B0015
  • 5) K331033-K331035
  • All parts assembled into pSB1K3
White colonies were picked from the plates and used for overnight cultures.

Saturday

Cells from the overnight culture (May 20, 2011) were mini-preped. Then mini-preped samples were restricted and ran on a 1% agarose gel.

Sunday

Express BamH1 restriction endonuclease in E. coli DH5α in comparison to wild type E. coli DH5α that were not induced. BamH1 restriction endonuclease was induced with arabinose. Optical density measurements were taken and a growth curve of both cultures was generated. Also samples after induction were taken to run on an SDS-PAGE.

Week 4 (May 23-29)

Monday

Justin recuperated after working through the weekend.

Tuesday

A 15% SDS-PAGE of samples taken from the BamH1 over-expression was ran. Gel looks promising ☺

Wednesday

Parts that were constructed recently were PCR amplified.

Thursday

An agarose gel was ran of the PCR products to determine which parts had the correct size. Parts that had an appropriate size range were sent for sequencing. Parts sent for sequencing:

  • 1) ROO10-BOO34
  • 2) K249002-BOO15
  • 3) K331033-K331035

Friday

Justin assisted team #1

Saturday

Time was spent researching, and assisting teams that spent the weekend working in lab.

Week 5 (May 30-31 and June 1-5)

Monday

Colones were picked from Morgan's (group 2) plates from 05/27/11 and set up for an overnight culture.

Tuesday

Overnight cultures from May 30, 2011 were mini-preped using the QIAGEN spin column method. Then the DNA was PCR amplified and ran on a 1% agarose gel. Parts that were sent for sequencing were:

  • 1) pBAD-P0440
  • 2) K249002-B0015

Assembly of 3 BioBricks!

  • 1) pBad-rbs + lumazine-dt
  • 2) K331033 + K331035
  • 3) R0010 + B0034
    • --> We want to have pLacU-rbs-lumazine-dt, but since we have pbad and rbs we will use above. This will result in expressing lumazine synthase ASAP :)

Dustin helped out with construction of parts

Wednesday

Justin helped out team 3

Thursday

Justin figured out what we need to order for lab, and compiled sequencing results.

Set up a PCR of Assemblies from May 31 to run on an agarose gel of constructs.

Friday

Ran PCR products from June 2 on an agarose gel and retransformed DNA in DH5α

Transform DNA that will be sent for sequencing on Monday (suspected BioBricks). Parts that will be transformed:

  • 1) K249002 + B0015
  • 2) pBAD + P0040
  • 3) R0010 + B0015
  • 4) K249002 + B0015
  • 5) K331033 + K331035

Saturday

All plates from transformations had some white colonies. Ranged from a lawn of white colonies to very few white colonies. White colonies were picked off each plate and a overnight culture was setup.

Sunday

Dee ran an agarose gel of several parts (Ryan pulled these out of glycerol stocks) to determine which parts should be sent for sequencing.

Week 6 (June 6-12 )

Monday

Cleaning the lab up a bit. So much work on our project has been done so far. Go team!

Tuesday

Helping Ryan figure out some PCR issues

Wednesday

Reading synthetic biology papers and preparing for SynBio 5.0! Also getting some autoclaving done.


Friday

Attending Journal club at the University of Lethbridge. Got some great ideas

Saturday

Mini-preped 5 samples/ colonies that Nathan assembled (pBAD-rbs-lumazine-dT) using QIAGEN mini-prep method. Then the parts were PCR amplified, and will later be restricted and ran on a agarose gel.

Sunday

PCR products from June 11, 2011 were ran on a 1.5% agarose gel to confirm their size

Week 7 (June 13- 19)

Justin left for ISMOS-3 on June 12, 2011. Had a great time on June 13,2011 listening to the keynote Steve Larter for the University of Calgary on "Microbes or Mass Transport -What are the Real Barriers to Production Scale Microbial Gasification of Oil or Coal to Produce Methane or Hydrogen in Subsurface Reservoirs?" The conference was very informative applied microbiology and molecular biology research that is occurring in the oil sands.

Then Justin left for SynBio 5.0 held at Stanford University. "I Attended SynBio 5.0, an international conference held this year at Stanford University in Palo Alto, California. At this conference, fellow team members and I got to see some of the leading experts in the field of synthetic biology and some of the amazingly creative research they are doing. Considering my love for iGEM, this conference gave me a whole new passion for this years iGEM competition." - Justin Vigar (transcribed by Harland Brandon)

Week 8 (June 20-26)

Monday

Planed out experimental protocol for over-expressing CFP with an agranine (10) tag (K331033) and purification using cation exchange

Tuesday

Gathered materials needed for over-expresion and purification of CFP with an arg tag. Made buffers for purification.

Wednesday

Setup overnight culture of CFP with arg tag (K331033) for over-expression.

Thursday

Over-expressed CFP with arg tag (K331033). Cell were frozen for purification down the road.

Week 9 (June 26-30 )

Monday

Calibrating pipettes, filling tip boxes and autoclaving. And mopping floor, Woo!

Tuesday

Aided team # 2

Wednesday

PCR amplified

  • 1) lumazine synthase-dt from glycerol stocks
  • 2) R0010-B0034 assembly
  • 3) R0010 in pSB1A2 (control)
  • 4) K331035
  • 5) K331033
  • 6) pSB1A3 (J04450)
  • 7) lumazine synthase

Then Ran PCR products on a 2% agorose gel.

constructs that were the correct size were:

  • 1) Lumazine synthase
  • 2) lumazine synthase-dt
    • the rest were undetermined.

Thursday

Attempted purification of CFP arg (K331033) tagged protein. There are some issues that need to be solved.

Friday

Problem solving and working on wiki.

Week 10 (July 5-10)

Tuesday

Clone J04500, pBAD-B0034, and K346007 into E. coli DH5α

Wednesday

Pick colonies of cloned parts, July 6, 2011, and started overnight culture

Thursday

Express BamH1 and show that growth is hindered; Minipreped cell cultures that were grown overnight to determine quality/quantity/size of DNA of certain parts; Assebled pLacI-RBS with lumazine-dT. Also a growth curved of a induced (arabinose) and un-induced culture were generated from optical density measurements.

After 8 hours dilutions were made from the induced and un-induced cultures and plated.

Results

Friday

Mini-prep cell cultures that were grown overnight, by Nathan, to determine the quality/quaintly/ size of DNA of parts:

  • 1) J04500
  • 2) pBAD-B0034
  • 3) K346007

Mini-preped samples were restricted then ran on a 1% agarose gel. We were unable to detect DNA for pBAD-B0034 on the agarose gel. All other parts were visible and had an acceptable size.

Saturday

Continue assembly on our lumazine BioBrick, which I'm really excited about. I think we will be able to construct our whole massive construct with lumazine synthase and the fluorescent proteins. It will be so cool if we can show that the construct will express the proteins and work as a true interchangeable part!!!

Assembly of pLacI-rbs (J04500) with lumazine.

Sunday

Transformation of yesterdays work! Transformed lumazine construct into E. coli DH5α.

Week 12 (July 17-24)

Wednesday

Determine if xyl transformations were successful and if K346007 was successfully transferred from pSB1C3 to pSB1K3. This was done by restricting the mini-preps and running a 1% agarose gel. It looks like we might have successfully assembled for the gel:

  • pSB1A3 promotor-rbs-xylJ-dt
  • pSB1A3 promoter-rbs-xylE-dt
  • pSB1A3 promoter-rbs-xylF
  • pSB1A3 promoter-rbs-xylk
  • pSB1A3 promoter-rbs-xylQ
  • pSB1A3 promoter-rbs-xylE-pSB1A3 promoter-rbs-xylQ
  • pSB1A3 promoter-rbs-xylE-pSB1A3 promoter-rbs-xylF
  • pSB1A3 promoter-rbs-xylE-pSB1A3 promoter-rbs-xylJ
  • pSB1A3 promoter-rbs-xylE-pSB1A3 promoter-rbs-xylK
  • J04450 pSB1C3
  • J04450 psb1k3
  • K346007 psB1K3
These will need to be sequence to confirm they are the parts we believe them to be.

Thursday

Today Team Justin determined if the assembly of R0010 (placI-rbs) and IC346007 (Agn43) was successful. Note: Boris did this assembly 07/20/11, see team 1 book for details. Plates that were transformed with parts do have white colonies :). These white colonies were selected and colony PCR was performed using the Prefix and Suffix primers.

Friday

Samples from colony PCR (yesterday) were ran on a 1% agarose gel. Amplification was successful! However, no inserts of right size are present.

Week 13 (July 25-31)

Monday

Assemble K331035-K331033 and R0010-luma-dT

First what happened is the BioBricks were restricted out of the plasmids. Then to determine the concentration of DNA after the restrictions a 1% agarose gel was ran.

The team has made so much progress, we are all very excited for Indianapolis! We had such a successful team meeting:) GO TEAM!

Tuesday

Determine DNA concentration of J04500, K331033, K331035, and lumazine synthase dt. Team JV (Justin and Mr. Vigar) concluded that DNA concentration was too low for the spectrophotometer to read. However, the DNA was still assembled and transformed (by Ben).

Then K331033 was ligated with K331035, and J04500 with lumazine-dt. Constructs were transformed into E. coli DH5α cells

Wednesday

Colonies were counted from yesterdays transformation. Both constructs resulted in white colonies --> We'll perform colony PCR on these samples see July 27, 2011


Week 14 (August 1-7)

Tuesday

First day in a three day experiment to isolate enhanced lumazine synthase from E. coli BL21(DE3) cells. Set up overnight culture, checked plasmids, made buffers.

Wednesday

Second day, lumazine synthase is overexpressed. Open cells and run SDS-PAGE gel. Purification of lumazine synthase.


Saturday

Concentrated lumazine synthase. SDS-PAGE done to confirm this.

Week 15 (August 8-14)

Monday

Buffers made for chromatography. Two assemblies are ran for lumazine + inverter and inverter + tagged fluorescent proteins.

Friday

Colony PCR of assemblies. Confirm inverter + tagged fluorescent.

Week 16 (August 15-21)

Tuesday

Picked colonies from plates to get more plasmids to assemble with.

Week 17 (August 22-28)

Tuesday

Assemblies of lumazine + inverter and fluorescent proteins.

Wednesday

Transformation of yesterdays assemblies.

Thursday

Helped out team 2 while Ryan is gone by testing assemblies of mms6 with polypetide signal tags and working with Dustin to assemble xylE with arginine polypeptide signal tag.

Friday

Mms6 testes negative on an agarose gel.

Week 18 (August 29 - September 4)

Monday

Prepping cellular samples for electron micrograph imaging.

Tuesday

Finished prepping samples for electron micrograph imaging.

Friday

Images of cells expressing lumazine synthase taken.


Week 20 (September 12-18)

Tuesday

LB cultures made from glycerol stocks.

Wednesday

Mini-prepped cultures and assembled the isolated parts into psb1c3.

Thursday

Continued assemblies from Wednesday.

Friday

Continued assemblies from Wednesday and Thursday.

Week 21 (September 19-25)

Prepared for aGEM in Edmonton

Week 22 (September 26-28)

Monday

Sent parts to the registry.

Tuesday

Sent parts to the registry with Ryan.

Wednesday

Gone to Calgary to give a presentation on the synthetic biology and the future of the oil sands.
Retrieved from "http://2011.igem.org/Team:Lethbridge/Notebook/Lab_Work/Justin"