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Team:Paris Bettencourt/Notebook/Drug Screening/Friday 5th July.html
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Latest revision as of 09:43, 13 August 2013
Drug Screening
ASDFFriday 5th July
Planning transformations for the next week
We decided to partly reproduce Figure 3C from: http://www.jbioleng.org/content/5/1/7:
So, we would pursue to grow the following strains on a minimal media M9 plate, supplemented with all amino acids
aside from the sulfur containing ones, and sulfite ans the only sulfur source:
We already thought we have all of the strains we need, however, an overnight culture was launched of a few single colonies from this double plasmid strain (sD001+pD004+pD005, which supposedly had all 3 genes SIR FNR and Fdx) and it didn't grow.
This most likely occurred because the strains didn't have Spec antibiotics resistance, as the other spec resistant strain containing
the FNR gene (pD004), was found not to be resistant as well.
Illustration
1: Strains will be grown on a minimal media M9 plate, supplemented
with all amino acids aside from the sulfur containing ones; plates
will have increased glucose, and sulfite as the only sulfur source.
Growth is expected only for WT and the strain containing all three
alternative genes of sulfur reduction.
Therefore, we would re-make:
sD001+pD004+pD005 E.coli:ΔcysI, Δfpr, ΔydbK:: soFD, zmSIR ;Chloramphenicol ,zmFNR; Spectinomycin- with all three genes.
sD001+pD004: E.coli:ΔcysI, Δfpr, ΔydbK:: zmFNR; Spectinomycin
and make
sD001+pD004+pD006 E.coli: ΔcysI, Δfpr, ΔydbK::zmFNR; Spectinomycin, zmSIR; Chloramphenicol
These transformations will be done by using two strains of competent cells prepared using Protocol 2 :
sD001 competent cells were transformed with:
pD004
Double transformation: pD005 +pD004
sD007 (sD001+pD005) competent cells were transformed with:
pD004
These transformations will be done by heat shock using protocol 1.
