Team:British Columbia/Notebook

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June 5th

Grew cell cultures of EPI 300, BL21, and DH5α, with plasmid PIJ 790 in them, harvested, and stored at -80 freezer.

-Ruichen

June 6th

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 KAN plates with control.

-Ruichen

June 7th

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 antibiotics of Amp (10µg/ml) and Chlor (34µg/ml), 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 8th

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:

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 9th

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