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Team:Lethbridge/Notebook/Lab Work/Justin
From 2011.igem.org
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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:
Restrictions were done (standard iGEM restriction enzymes) and then the parts were ligated together.
- 1) pLacI-sRBS
- 2) pBAD-sRBS
- 3) K331033-K331035
- 4) K331025-B0015
- 5) R0040-K331029 into pSB1T3
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:
White colonies were picked from the plates and used for overnight cultures.
- 1) pBAD-P0440
- 2) R0010-B0034
- 3) K331025-B0015
- 4) K249002-B0015
- 5) K331033-K331035
- All parts assembled into pSB1K3
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.
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:
These will need to be sequence to confirm they are the parts we believe them to be.
- 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
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.
