Field of Science

Experiment plans

OK, my assignment (from the RA) is to test transformation of E. coli with nutritional markers rather that the crp::kan cassette she's used so far.

The first step is to inoculate strain W3110 into, say, 10 ml of LB broth and grow it overnight, and then tomorrow morning do a DNA prep. This will be the donor DNA for my transformations.

I'll be transforming two strains, C600 and BW25113. C600 is a work-horse strain from my grad-school days; genetically it is lacY (it can't take up lactose), thi (it needs the vitamin thiamine in its medium), and thr leu (needs the amino acids threonine and leucine in its medium). Its full genotype is F- tonA21 thi-1 thr-1 leuB6 lacY1 glnV44 rfbC1 fhuA1 λ-. F- and λ- means it differs from the ancestral K-12 strain in lacking the conjugative plasmid F and the lambda prophage. The tonA21 mutation removes a protein used as a receptor by phage T1, the glnV44 mutation (also called supE44) changes a glutamine tRNA gene so it inserts its glutamines at what would be UAG stop codons, rfbC catalyzes a step in synthesis of the outer-membrane sugar rhamnose, and fhuA helps cells take up ferrichrome (an iron-scavenging siderophore). I've listed all these because I want to make sure I've thought about all the factors that might confound my experiments - I see none here.

The other strain, BW25113, comes from Barry Wanner by way of the fantastic Keio group in Japan who have generated many of the E. coli clones and cassette mutants we've used. Its full genotype is F- ∆(araD-araB)567, ∆lacZ4787(::rrnB-3), lambda-, rph-1, ∆(rhaD-rhaB)568, hsdR514. So it has deletions of the araBAD operon (can't use arabinose) and the rhaBAD operon (can't take up rhamnose). rph-1 helps processing of tRNAs, and hsdR is a restriction nuclease that cuts DNA lacking a specific methylation. 

I'm only interested in the lacZ mutation; this is an insertion of 4 rrnB terminators that block transcription of the lac operon. I'd better check for an associated deletion, and for the size of the insertion, as these factors may influence the efficiency of recombination. Well, after quite a lot of searching I've come up with not much. This allele has an unspecified deletion and an insertion of 3 (or 4) copies of the rrnB transcriptional terminator in the lacZ promoter. I've emailed Barry Wanner asking for more details that would let me estimate the length of the heterology between this allele and a wildtype allele. (Prompt reply! The heterology is about 1 kb.)

What controls will I want to do? No DNA, to confirm that I'm seeing transformants and not new mutants. Cells without the inducing sxy gene (with a no-insert version of the plasmid). Cells with the sxy plasmid but without IPTG induction.

What will I select for? In BW25113 I can only select for Lac+, so I'll need minimal plates with lactose as well as minimal plates with glucose. BW25113 doesn't need any supplements. In C600 I can select for Lac+ by providing lactose as the only sugar, but the plates need to be supplemented with thiamine, threonine and leucine. I can also select for Thr+ and for Leu+ by not adding these amino acids to the medium.

So I'll need stocks of glucose and lactose (20%, I think - it's been a very long time since I did simple genetics in E. coli) and of threonine and leucine (10 mg/ml is standard for amino acids, I think). Thiamine I might as well put into all the agar. And minimal salts, and autoclaved agar.

Because I'll be growing up cells with both the inducing plasmid and a no-insert plasmid, I can also follow their growth in enough detail to see whether the insert slows growth.

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