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
Tuesday
Wednesday
Thursday
Friday
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. This was a mind blowing experience.
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.
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