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Team:Lethbridge/Notebook/Lab Work/Group3
From 2011.igem.org
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June
June 21st, 2011
Restriction, Ligation, and Transformation Lumazine synthase - double terminator and pBAD - RBS into pSB1C3
1. Restriction
- Restricted lumazine synthase - dt out of pSB1K3 and pBAD - RBS out of pSB1T3 with EcoR1 and Pst1
- Restricted destination plasmid pSB1C3 with EcoR1 and Pst1
2. Ligation
- Ligated lumazine synthase-dt into destination plasmid pSB1C3
- Ligated pBAD-RBS into destination plasmid pSB1C3
- Transformed lumazine synthase - dt in pSB1C3 into DH5∝ E. coli cells
- Transformed pBAD-RBS in pSB1C3 into DH5∝ E. coli cells
June 24th, 2011
Picked cells from June 21st transformation
June 25th, 2011
Minipreped Cells picked on June 24th, 2011
June 28th, 2011
Restriction of June 25th miniprep and ran on agarose gel
Lumazine - dt construct successfully inserted into pSB1C3! :) Glycerol stocked.July
July 8th, 2011
Performed antibiotic assay on lumazine - dt in pSB1C3
Samples from one colony grew in Kanamicyn; this stock was discardedPCR:
- PCR'd out lumazine - dt
- Used VF2VR prefix and antisense suffix
Ran samples on agarose gel to confirm sizes
July 10th, 2011
PCR'd out lumazine synthase - dt from June 28th using VF2VR prefix and antisense suffix
Ran on an agarose gel for 45min at 70volts
July 25th, 2011
Today we analyzed 4 of Team 2's DNA samples. To do this we ran a restriction digestion reaction cutting the biobrick out of the plasmid. We then ran these digests on an agarose gel. The gel looked nice and we gave it to Team 2 for further analysis. They owe us one!
July 28th, 2011
Colony PCR with VF2VR prefix and antisense suffix:
k331033-k331035 in pSB1C3
P0440-K331033 in pSB1A2
J04500-lumazine synthase-dt in pSB1AK3
pBAD-P0440 in pSB1A2
lumazine synthase-dt-pBAD in pSB1A2
July 29th, 2011
For control comparision of parts from July 28th PCR we ran a PCR of:
K331035
K331033
Lumazine synthase - dt
P0440
Control
Ran PCR with VF2VR prefix and antisense suffix samples and controls from July 28th on an agarose gel. PCR was successful!
Additionally, we did an EcoRI restriction of:
J04500-Agn 43 sample 1
J04500 -Agn 43 Sample 2
J04500 - Lumazine synthase - Dt sample 3
K346007
Lumazine synthase - Dt
We then ran samples on an agarose gel to confirm sizes
August
August 2nd, 2011
Transfer of enhanced lumazine synthase from pET-28a into pSB1C3
Restricted with EcoR1 and Pst1
Ran on 1% TAE agarose gel for 90 minutes at 80V
- Gel melted -- unsuccessful.
Picked colonies to be glycerol stocked from successful colony PCR from July 28th: k331033-k331035 in pSB1C3 P0440-K331033 in pSB1A2 J04500-lumazine synthase-dt in pSB1AK3 pBAD-P0440 in pSB1A2 lumazine synthase-dt-pBAD in pSB1A2
August 4th, 2011
- Enhanced lumazine synthase restricted with EcoR1 and Pst1
- pSB1C3 restricted with EcoR1 and Pst1
- pBAD - P0440 restricted with EcoR1 and Pst1
- lumazine - dt-pBAD -RBS restricted with EcoR1 and Pst1
Gel ran at 80V for 90 minutes and a gel extraction was performed
- Enhanced lumazine with pSB1C3
- pBAD - p0440 with pSB1C3
- lumazine - dt -pBAD - RBS with pSB1C3
- Transformation of DNA into competent DH5∝ E. coli cells
August 5th, 2011
Transfer of p0440 - K331033 from pSB1A2 into pSB1C3
- p0440 - K331033 restricted with EcoR1 and Pst1
Ran on 1% TAE agarose gel for 90 minutes at 80V
- Unsuccessful restriction :(
August 6th, 2011
Transfer of p0440 - K331033 from pSB1A2 into pSB1C3
- p0440 - K331033 restricted with EcoR1 and Pst1
Ran on 1% TAE agarose gel for 90 minutes at 80V
- Gel melted -- unsuccessful TAE suspected to be the issue
- J04500 and P04400-K331033 were restricted with EcoRI and SpeI, Lumazine synthase-Dt-pBAD and K331035 were restricted with EcoR1 and SpeI
Ran Restrictions on 1X TAE agarose gel
- 8 samples ran total, 2 of each restriction were done, and run in separate lanes, P0440-k331033 showed not bands on Gel
Performed a successful Gel extraction
August 7th, 2011
- J04500 and lumazine synthase- dt- pBAD in pSB1A2
- Controls ran using lumazine- dt- pBAD and 2 microlitres dH20
August 8th, 2011
Performed a miniprep of August 4th transformation, pBAD+p0440 and lumazine synthase-dt+pBAD
Ran a 1% TBE agarose gel Results of Gel look good! :)
Transformation of Enhanced lumazine synthase in pSB1C3 and plated
Picked colonies for tomorrow:
- J04500 Lumazine synthase Dt in pSB1AK
- Lumazine synthase - dt- pBAD in pSB1A2
- P0440 - K331033 in pSB1A2
August 9th, 2011
Religated Enhanced lumazine synthase into pSB1C3
Transformation of enhanced lumazine synthase into competent Dh5alpha E. coli cells
[Team:Lethbridge/Notebook/Common_Protocols#Miniprep_Protocol|Miniprep]] of overnight cultures from August 8th (yesterday).
Restriction of:
- J04500 - Lumazine-Dt out of pSB1AK3 with EcoR1 and PstI
- P0440 K331033 out of pSB1A2 with EcoR1 and PstI
Ran 1X TAE agarose gel for gel extraction
- Gel ran at 80Volts for 60 minutes
- Results of gel analysis show peices at wrong sizes, potential restriction issues.
Picked colonies from August 8th plating of enhanced lumazine synthase for overnight culture.
August 10th, 2011
Miniprep'd Enhanced lumazine from over night culture on August 9th.
Ligated together
- K331033 - K331035 + pBAD - P0440 in pSB1C3
- I13453- P0440 + Lumazine synthase Construct in pSB1A2
- Note: Ligation used 1.0 Micro liters of T4 Ligase in each ligation instead of 0.5 Micro liters
Transformation of ligation mix
August 11th, 2011
Picked colonies from glycerol stocks:
- P0440 - K331033 in pSB1A2
- pBAD - P0440 in pSB1A2
- Lumazine synthase - dt - pBAD in pSB1A2
- J04500 - Agn 43 in pSB1AK3
Agarose gel and gel extraction of samples from August 9th:
- J04500 - Lumazine synthase - dt
- P0440 - K331033
Extracted parts ligated into pSB1C3
Control Ligation performed with restricted pSB1C3 and dH20Transformation of ligation mixture.
- (No colonies ended up growing)
August 16th, 2011
Repeated of August 11th ligations and transformations of:
- J04500 - Lumazine synthase - Dt
- P0440 - K331033
August 28th, 2011
Preparation of cells for Transmission Electron Microscopy (August 29th to Sept 1st)
DH5α Cells containing BBa_K542008 as well as controls containing only plasmid pSB1C3 were grown overnight at 37°C.
August 29th, 2011
Approximately 400μL of the cells that were picked Aug. 28th were resuspended in 5mL SOC media, and induced with 5μL or 10μL of 1M IPTG. Cells were again incubated at 37°C for approximately 4 hours. Cells were then spun down at 6000xg for 2 minutes.
Cells were resuspended in 500μL gluteraldehyde/formaldehyde fixative and incubated at room temperature for 2 hours. The solution was then spun at 6000xg for 2 minutes.
Cell pellet was resuspended in 0.1 sodium cacodylate buffer and incubated for 10 minutes. Solution was centrifuged for 2 minutes at 6000xg. This step was repeated an additional 2 times. Cells were then resuspended in 1% osmium tetroxide fixative in 0.1M CaC buffer and incubated at room temperature for 1 hour. Solution was centrifuged at 6000xg for 2 minutes. Cells were then resuspended in Milli-Q H2O.
The cells were spun down at 10000xg for 5 minutes, and resuspended in 4% BIO-RAD standard low melting temperature agarose at 60°C. A drop of this cell-agarose mixture was then added to slides in order to harden, and incubated in Milli-Q H2O overnight.
August 30th, 2011
Agarose-cell mixtures were cut into cubes of approximately 1mm x 1mm x 1mm.
They were incubated in increasing alcohol concentrations:
- 50% - 5min
- 70% - 10 min
- 80% - 10 min
- 90% - 10 min
- 95% - 2 x 10 min
- 100% - 2 x 10 min
- Abs 100% - 3 x 10 min
Then increasing concentrations of resin on rotator:
- 25% - 1 hr
- 50% - 1 hr
- 75% - 2 hr
- 100% - 15 min
- 100% - overnight
The cubes were left in 100% resin overnight on rotator.
August 31st, 2011
The cubes from the 100% resin were then placed in tubes with fresh resin and left in an oven for 24 hours to harden.
September
September 1st, 2011
The resin-embedded cubes of cells were then thin sectioned and placed on a carbon grid. They were stained with uranyl acetate and then positively stained with lead citrate then viewed under transmission electron microscope with the help of Doug Bray.
September 8th, 2011
- J04500 in pSB1AK3 SpeI and PstI
- Enhanced lumazine synthase-Dt in pSB1C3: XbaI and PstI
- J04500-Enhanced lumazine synthase in pSB1AK3: EcoRI and SpeI
- Dt in psb1AK3: EcoRI and XbaI
- Enhanced lumazine synthase in pSB1C3: EcoRI and SpeI
- Dt in pSB1A2: EcoRI and SpeI
- Restriction mixture into freezer overnight after 1 hour at 37 Degrees C
September 9th, 2011
Ran agarose gel with samples from September 8th.
- Note: more issues with the Gels, Volts too low, Amp too high, Assistance from Weiden lab PhD student, explains in depth how gel rigs, power packs and agarose all work, Helped trouble shoot each area, which lead to Changing the 1X TAE from weiden lab stock. This gave constant Voltage and lowered the amps, since the agarose gel was made from our TAE, gel had to be run at constant amps for several hours to prevent it from melting.
Gel extraction went well! All pieces showed up with the exception of J04500-Enhanced lumazine in pSB1AK3
Ligations of
- J04500 and Enhanced lumazine synthase-Dt in pSB1C3
- Enhanced lumazine synthase and Dt in pSB1AK3
- Enhanced lumazine synthase and Dt in pSB1A2
September 10th, 2011
Assembly of Ptet-Rbs in pSB1A2 to have XylE- Arg Tag-dt
Restriction of:
- pTet-rbs with SpeI and PstI
- XylE Atag- dt with XbaI and PstI
While samples restricted at 37 degrees Celcus for 1 hour, we made a 1% TAE agarose gel using TAE from Weiden stocks for both Gel and buffer.
Agarose gel ran at 80Volts for 60 minutes. Was very happy to see the gel running at normal Amps and reaching regular Volts, No more melting Gels!!
Gel analysis by UV indicated restrictions did not work for XylE
September 11th, 2011
3 days back and nothing has worked, persistence is the only way we're going to get through this!!
Built a new copy of Xyle + S04261 since the stock copy is unaccounted for.
Found prerestricted Xyle and S04261 in team 1 book, and located in freezer, performed an overnight ligation Restricted Enhansed Lumazine Synthase-Dt in pSB1C3 cut at EcoRI and XbaI to later ligate with previously restricted J04500 cut at EcoRI and SpeI
- J04500 out of pSB1AK3 with EcoRI and SpeI (to be ligated with the Enhanced lunazine synthase-dt in pSB1C3)
- Enhanced lumazine synthase in pSB1C3 with SpeI and PstI
- dt D6 was restricted at XbaI and PstI. (later to learned I should have left dt in plasmid and removed the enhanced lumazine synthase, Dt was too small and ran off gel.)
Ran all parts on a 1% TAE agarose gel
- Enhanced lumazine synthase-dt, J04500, and Enhanced lumazine synthase in pSB1C3 all restricted well, but dt D6 did not as it was too small and ran off the gel.
Ligation of each of the Enhanced lumazine synthase-dt with the gel extracted J04500 and the previously restricted J04500
September 12th, 2011
Transformed and plated cells from ligation mixture from September 11th
September 13th,2011
End of the first Week worth of classes, and still nothing grows/ works Why?? I found out that I am using the wrong enzymes, a type that wont heat kill (Later questioned, since I'm running a gel for extraction. I was informed the heat kill process doesn’t need to be done when running a gel for extraction when I was introduced to the gel extractions, I asked why are we doing it this way, the answer was ‘It’s more efficient!’ apparently in theory only)
Ok one More Kick at it, New enzymes check, Proper TAE check, advice from Phd students Check, Objective: Get something to work! Feeling I have it all figured now, I set forth once more.
Restriction of:
- J04500 in pSB1AK3 with at SpeI and PstI
- Enhanced lumazine synthase- Dt with at EcoR1 and PstI
- J04500-Enhanced lumazine synthase with EcoRI and SpeI
- dt D7 in pSB1AK3 with EcoRI and XbaI
- Enhanced lumazine synthase with EcoRI and SpeI
- dt D6 in pSB1A2 Cutting with EcoRI and XbaI
- pTet-Rbs in pSB1A2 with at SpeI and PstI
- XylE-ATag-Dt with XbaI and PstI
Made Gel while Restrictions incubated,
- 1% TAE Agarose Gel then stored in fridge overnight, Note: don’t store on top shelf, Gel will freeze
Stored restricted DNA in Freezer overnight to be used in gel in the morning,
September 14th, 2011
Gel froze overnight in fridge, so made two new 1% TAE agarose gels (one small one large) ( so much for loading a gel and going to class!)
- Ran gels at 80Volts for 90 minutes
Results:
- Enhanced lumazine synthase –dt did not work (restriction issue?)
- J04500 –Enhanced lumazine synthase did not work(restriction issues?)
- XylE –ATag-dt was not on gel (portion too small for successful gel extraction/ not enough lanes)
Only Enhanced lumazine synthase and dt D6 in pSB1A2 were available to proceed to gel extraction,
Gel Extraction Unsuccessful Pack up go home try again in a few days
September 21st, 2011
Cells containing the BBa_K542008 construct as well as controls containing pSB1C3 were grown overnight at 37°C.
September 22nd, 2011
Cells grown on September 21st, 2011 were spun down at 10,000rpm and then resuspended in 1X phosphate buffered saline (PBS). The solution was centrifuged at 10,000rpm, decanted and resuspended in 4% paraformaldehyde (PFA) and left overnight.
September 23rd, 2011
The 4%PFA-cell solution was then centrifuged at 10,000rpm and cells were resuspended in glycerol. The glycerol-cell mixtures were then placed on slides, coverslipped and sealed.
The slides were viewed with an Olympus FV1000 spectral confocal at 60X magnification.
