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Team:British Columbia/Notebook
From 2012.igem.org
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May 28
Started making first batch of DH5α and EPI300 competent cells. See Competent Cell Production.
May 30
Finished production of the competent cell protocol that was initiated on May 28. OD_600 values were monitored as cells grew until when it reached 0.420 for DH5α and 0.443 for EPI300, upon which they were iced immediately. Competent cells were not flash-frozen with liquid nitrogen, but were placed directly into the -80ºC freezer.
Made 2 bottles of 400 mL M9 + glucose media.
June 5
Made EPI 300, BL21, and DH5α competent cells with plasmid pIJ790 in them.
-Ruichen
June 6
Learned how to electrophorese cells with biobricks (1-7B, 2-17F, 2-14N, 2-9B, 3-13M, 3-14E, 4-3I, 5-1A, 5-12O), and plated them on plates to grow colonies.
Tested kanamycin (Kan) plates with control.
-Ruichen
June 7
Retreated the plates previously spread, KAN plates worked and most of them grew into colonies or covered plates completely. (advised to spread the plates with 50µl recovered cells instead 100µl in the future for better colony identification).
Made more 10µg/ml ampicillin (Amp) and 34µg/ml chloramphenicol (Chlor), and more agar plates of Amp and Chlor (half bagful respectively).
Electrophoresed 1-1N, 2-2K, 1-5P, 2-21O using DH5α, and plated them.
-Ruichen
In the afternoon, we (Ruichen, Mehul, Grace) made many more K12 competent cells, which we put into storage. (The protocol is now on the wiki.) There was also talk from Jacob and John about buying $0.50 worth of gasoline/diesel from the nearest gas station to test solvent resistance of some of our cells. I left before that happened, though. (Would have been fun to see the attendant's reaction!)
- grace
June 8
Tried to make more EPI300 cells, though no obvious growth even after hours for the second time, and decided to leave it over night to see if there are any changes.
Electrophoresed K12 with PIJ 790 plasmid, and plated them on Chlor plates with control plates to examine the plates.
- Ruichen
Prepared a 96 well plate for co-culture experiments with the the following ratios:
| GFP | RFP | YFP | 0.5G/0.5R | O.67G/0.33R | 0.33G/0.67R | 0.5G/0.5Y | O.67G/0.33Y | 0.33G/0.67Y | 0.5R/0.5Y | O.67R/0.33Y | 0.33R/0.67Y | ||
| GFP | RFP | YFP | 0.5G/0.5R | O.67G/0.33R | 0.33G/0.67R | 0.5G/0.5Y | O.67G/0.33Y | 0.33G/0.67Y | 0.5R/0.5Y | O.67R/0.33Y | 0.33R/0.67Y | ||
| GFP | RFP | YFP | 0.5G/0.5R | O.67G/0.33R | 0.33G/0.67R | 0.5G/0.5Y | O.67G/0.33Y | 0.33G/0.67Y | 0.5R/0.5Y | O.67R/0.33Y | 0.33R/0.67Y | ||
| 0.33G,R,Y | 0.33G,R,Y | 0.33G,R,Y | |||||||||||
| Media | Media | Media |
All cells were derived from EPI300, and were grown in M9 media to allow for accurate OD measurements. a total of 10 uL culture in each case was added to 190 uL M9 media.
However, the plate reader was later found to be broken. The plate was placed in the 4° cold room until such time that the experiment could be done.
The wells were plated by GFP first, then RFP, the YFP. There was a slight accident that involved losing some of the YFP culture, but enough was recovered to fill the plates. It is likely that the GFP cultures spent over 30 minutes in the plate before the YFP was added, so some readings may not accurately reflect the ratios shown above.
-Jacob
June 9
The EPI300 did grow!
- Ruichen
Bought $1 of 91 octane (no ethanol) gas from the Shell on 10th. It's in a red jerrycan in the flammables cabinet near our bench.
JohnHenry 14:31, 9 June 2012 (CDT)
Experiment plan for week of June 10-16th:
1) Kill Switch Assay for Colicin E7, Colicin E2, BamH1, H2O2
Colicin E7: There is currently a plate of K12 cells with protein immunity and Colicin E7. Lysis protein (cuts the immunity gene to allow for Colicin E7 production) will be transformed into K12 (Sat). Cultures will be set up for both the protein immunity+Colicin E7 and the Lysis (Sun). The two cultures will be mixed and grown up to correct OD then plated (Mon). Plates will be checked for growth (Tues).
Colicin E2, BamH1, H2O2: There are currently plates of K12 cells with Colicin E2 and H2O2s. Colonies will be picked and cultures will be set up for each of these (Sat). The cultures will be grown to correct OD and induced then plated (Sun). Colicin E2 is induced low concentration of ampicillin, BamH1 is induced with arabinose, and H2O2 is induced with 2nM of AHL. Plates will be checked for growth (Mon). BamH1 will be transformed into K12 cells and plated (Sat). Same steps will be taken as above and plates will be checked for growth (Tues).
2) Solvent Tolerance Assay
Cultures of K12 cells with 3I plasmid will be set up (9am on Sun) in gasoline and pentane. OD will be checked every hour for 12 hours or less until stable phase (Sun). Protocol is on the TU Delft 2010 page [1].
3) PCR RFP, YFP, GFP on to pcc1 fosmid
There will be a PCR tutorial on Thursday 6pm with the primers for putting the RFP, YFP, and GFP from the biobrick on to pcc1 fosmids.
- Marianne
Wanted to start the solvent tolerance assay today, but previous protocols required 12 hours and by the time we got the gasoline, it was already 12. We decided to wait a day. In the mean time, we started some cultures and plates for a few kill switches that could also be assayed. The cultures were of a H2O2 producing strain, and another strain that expressed the colicin E2 operon, which is inducible under low (10 uG/mL) concentrations of B-lactam antibiotics, such as ampicillin (http://www.sciencemag.org/content/305/5690/1629.full#F2). Despite documentation of plating of the BAMHI kil switch, the plates were unable to be found, so another transformation was done, along with a transformation for the lysis gene from the colicin E7 operon. The colicin E7 toxin and immunity protein have already been grown in cultures. The tentative plan is to see if the two cultures interact by the lysis gene cleaving the E7/immunity complex, causing cell death. There was no one who knew how to use the plate reader in the lab today, so the co-culture experiments were further put on hold. It should be noted that the kill switches, unless otherwise noted, have been transformed into K12 E. coli cells, which are RecA positive, allowing the SOS promoter to work.
- Jacob
June 11
Started kill switch assays on colicin E2 and BamHI. Colicin E2 is on the SOS promoter, which is best switched on by mitomycin. However, we currently do not have access to mitomycin or any of its closely related derivatives, so, judging by the previous;y mentioned paper, it might be able to be induced by b-lactam antibiotics in low concentrations.
Procedure outline: Dilute 1:100 22A, 3C and wt strains in 25 mL LB. Measure OD every hour. At start of exponential phase, add arabinose to 0.01% or amp to 4 ug/mL to strain+control. Plate 25 uL 2 hours later, mid exponential phase. Details to follow.
The plate reader is still broken, with no estimated repair date.
-Jacob
Transformed biobricks 7C, 22C, and 20K into K12, 8G into BL21 pIJ790, 7C into EPI300 pIJ790, and 14C into DH5α pIJ790 for miniprep. After recovery, the cells were plated in the following manner: 22C and 20K on LB + Kan, 14C and 8G on LB + Amp, and 7C (both the K12 and EPI300 cells) on LB + Chlor. Different cells were chosen to test out the newly made competent cells. Only the K12 cells have been verified. 7C was transformed into K12 after, in addition to EPI300 pIJ790, because the 7C biobrick has Chlor resistance, which mimics the marker on pIJ790.
Quantified previously miniprepped biobricks using the nanodrop spectrophotometer.
June 12
Checked plates from Jacob's June 11 kill switch assay. Every single plate (3C+, 3C-, 22A+, and 22A- in both 5 uL and 10 uL plating quantities) grew a lawn. Only 3C+ and 3C- plates had a few distinguishable colonies.
Checked plates of the cells transformed on June 11 by Ting-Chia and started 3 mL cultures for miniprepping. Only the DH5α pIJ790 and EPI300 pIJ790 transformed with the 14C and 7C biobricks, respectively, did not seem show any growth upon the first check. It was unclear as to whether there were actually any 7C (EPI300 pIJ790) colonies growing, so both plates were returned to the 37ºC room to incubate. For the other plates that did show growth, single colonies were picked and inoculated in 3 mL starter cultures for miniprep. Cells were incubated at 37ºC at first, then at 30ºC to grow overnight.
QIAprep Miniprep kit from Qiagen was used to miniprep 4-3I, 2-3C, and 3-22A. The cells used were previously inoculated cultures that were stored at 4ºC. DNA product was stored in sterile dH2O and frozen in the -20ºC freezer.
We received plates of Rhodococcus sp. strain JVH1, Rhodococcus rhodochrous IGTS8 (ATCC 53968), and Baillus sphaericus IGTS9 (ATCC 53969) from Dr. Jonathan Van Hamme of Thompson Rivers University today. Single colonies were picked and inoculated in 3 mL LB at 37ºC at first, then at 30ºC to grow overnight. Plates were then parafilm-sealed and stored at 4ºC.
After 20 hours at 37ºC, the plates with 14C and 7C (EPI300 pIJ790) had colonies. 14C only had one colony, while 7C had a couple of small colonies. The origins of the colonies were uncertain, so the plates were discarded and the cells will be transformed in K12 the next day. Also, 13M biobrick (RFP gene), which has been verified, will be transformed into EPI300 pIJ790 and DH5α IJ790 to check if the competent cells are working properly or if they are to blame for the failed 14C and 7C plates.
