GS1783 E.coli strain大肠杆菌菌株BioVector NTCC质粒载体菌种细胞基因保藏中心

GS1783 大肠杆菌菌株

BioVector NTCC质粒载体菌种细胞基因保藏中心

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