Wet Lab Meeting
NEWS
- 2009 iGEM Plate has already arrived to Mexico, so we are waiting for it to be here this week.
- We need to do the Vibrio fischeri assay and competent cells.
- Luciferine and dodecanal were bought.
Done this week
- Zepeda: Trials with CI invertor of λ phage for LovTap, troubles with ligations, transformations, and restrictions.
- Jorge: Last week we decided to change the way to extract the Blue Promoter. We were using PCR, now we decided to synthesize an oligo containing the prefix to prime plasmid 30, the minimum blue promoter (50bp), and a restriction site for SpeI. The genomic DNA extraction performed by Miguel was checked by electrophoresis for both E. coli K12 and Vibrio Fischeri MJ11.
- Augusto: Working with Pcya h1, didn't come out well because at the moment of purification, it was lost. In order to continue we will need to wait for 2009 plate.
- Mariana: This week the ligation of luciferase with the double terminator was done, transformed and grown into E. coli strains. The plasmid extraction and its respective restriction with ECO RI and PST I has already been done and amplified by PCR.
Goals for the week
1. Luminiscence assay with Vibrio Fischeri, using a luminometer and the cameras provided by Alberto Soria. Check
how this assay is made (Protocols to grow the bacteria, incubation temperature, etc.).
2. Ask Claudia to talk to Alberto Soria and ask him for the cameras to make the Vibrio Fischeri luminiscence assay.
3. Usage of the BioBrick Promoter Measurement Kit to test the Blue Promoter. First, the promoter must have EcoRI and SpeI sticky ends, a GFP reporter with XbaI and PstI sticky ends and a backbone plasmid pSB3K3 with EcoRI and PstI sticky ends.
4. Ligation of the three parts (Blue Promoter, GFP reporter and Backbone plasmid).
5. Re-plate available FP's in LB medium (solid or liquid??) to make the fluorescence assay. The transfomation with the YFP reporter, will be used for fluorescence assay too. The transformation with GFP reporter and with the backbone plasmid will be inoculated in solid and liquid medium to make plasmid extraction. With
the extracted plasmid, there will be necessity for restrictions and ligations of them with the Blue Promoter to test its functionality.
6. For the Blue Promoter measurement with a GFP regulated under the constitutive promoter J23101, as a reference.
1. Do a PCR with Pfx for the punctual mutation on luciferase.
2. Ligate it into a plasmid with a constitutive promoter.
3. Ligate it into a plasmid for the mutation.
1. Help Jorge in the bioluminescence assay, mainly in the protocol search, as mention in Jorge's objective 1.
2. If competent cells are done for Wednesday, transformation of Cph8 (chromofore that receives red) with pcyA+Ho1, also pcyA+Ho1, Cph8.
3. Extract luxAB from Vibrio Fischeri MY11.
1. PCR to modify the RBS of GFP + luciferase.
2. PCR for the trpL + plasmid promoter.
3. Ligate GFP + inverter CI.
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