χρόνος πέρασμα June 29th 2011
- Transformation into E.coli DH5α of Pam Silver's plasmids.
- Last friday (29/06/2011) arrived the plasmids that Pamela Silver's lab sent to us. Today I transformed these plasmids into DH5α E. coli cells. The plasmids are in three 0.5 ml tubes and allegedly contained 5 μl of plasmid. Two of the three tubes did contain this quantity, but the one that contained the HydA and Fd genes only had approximately 1 μl of plasmid; Fernando Montaño and Miguel Ramírez advised me to dilute this last plasmid with TE 1:10. 9 μl of TE 1:10 pH 8 was added to the tube. The transformation was performed according to Miguel's protocol (50 μl of competent cells were used instead of 100 μl as the protocol says).
pPS3182 (Alias D9) contains commercially synthesized, yeast codon optimized HydEF and HydG maturation factors from Chlamydomonas reinhardtii in Biobrick modified pCDF Duet vector. The plasmid confers the host resistance to Spectinomycin.
pPS3186 (Alias E237). Expression vector with Clostridium acetobutylicum HydA + C-terminal strep-II tag in multiple cloning site 1 and 2[4Fe-4S] Ferredoxin in multiple cloning site 2 of BioBrick modified pET Duet vector. The plasmid confers the host resistance to Ampicilin.
pPS3183 (Alias D23) contains Desulfovibrio africanus PFOR in pACYC Duet vector. The plasmid confers the host resistance to Chloramphenicol.
- As the control construction we wish to build is supposed to serve as codon optimization control, the genes that come in plasmid pPS3182 are not usefull at all. Anyway, I transformed the three plasmids. The maturases wild-type sequences from C. reinhardtii are going to be obtained directly from the organism with PCR. I prepared 75 ml of LB solid medium with 75 μl of the respective antibiotic for each plasmid. Both Ampicillin and Spectinomycin were at a concentration of 100 μg/ml. Chloramphenicol was at a concentration of 25 μg/ml. I plated each plasmid in their respectives petri dishes and each had another petri dish non inoculated as a platting control.