Team:Paris Bettencourt/Notebook/Drug Screening/Friday 5th July.html

Drug Screening

Friday 5^th 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.