E.coli GS1783 strain

Description

E.coli GS1783 strain contains a chromosomal encoded, temperature-dependent
expression cassette for the recombination proteins and an
L-(+)-arabinose-inducible I-sceI gene to allow for convenient and highly
efficient mutagenesis without the use of additional plasmids.

The two-step Red-mediated recombination approach requires successive
transient expression of the Red recombination proteins (i.e., Exo, Beta, and
Gam λ phage proteins)
and the I-SceI endonuclease. The GS1783
strain is the preferred host for this procedure since it contains both a
heat-inducible promoter and an l-arabinose-inducible promoter, which control
the expression of the Red recombination proteins and the I-SceI endonuclease,
respectively.

Recovery

1.Obtain
an LB agar plate with the appropriate antibiotic.

2.Using
a sterile pipette tip, touch the bacteria growing within the punctured area of
the stab culture. (A sterilized wire loop or sterile toothpick can be used in
place of a sterile pipette tip.)

3.Run
this tip lightly over a section of the plate, as shown in the figure, to create
streak #1.

4.Using
another sterile pipette tip, pass through streak #1 and spread the bacteria
over a second section of the plate, to create streak #2.

5.Using
a third sterile pipette tip, pass through streak #2 and spread the bacteria
over the last section of the plate, to create streak #3.

6.Grow
overnight in a 37 ^o C incubator (unless a different growth
temperature is indicated on the plasmid datasheet).

7.In
the morning, single colonies should be visible. If the bacterial growth is too
dense, re-streak onto a new agar plate to obtain single colonies.