Part:BBa_K917002:Experience

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Applications of BBa_K917002

This part is designed for use in counter-selection experiments. Cells are grown in a high-sucrose medium (eg, on L-agar plates with 10% w/v sucrose). Only cells which have lost the cassette should grow. Literature suggests that this counter-selection cassette is prone to failure through loss-of-function mutations in sacB, which may occur at frequencies higher than loss of the cassette. To help distinguish such events, we have added the RFP from BBa-J04450, so that sacB loss of function will still produce red colonies, whereas true loss of the cassette leads to white colonies.

An quick preliminary trial was conducted using the modified version of this cassette, BBa_K917010, with BBa_P1001 kanamycin resistance cassette added. A mixed culture of E. coli JM100/pSBIC3-BBa_K917010 and a control strain expressing lacZ as a marker gene was grown overnight in L-broth. A 1e-6 dilution was prepared and 100 microlitres was plated to L-agar plates with chloramphenicol (40 mg/l), IPTG (90 mg/l) and X-gal (40 mg/l) (CIX plate) and to the same medium with 10% w/v sucrose incorporated (CIX-SUC plate). Colony counts were as follows:

CIX plate: 793 small blue colonies, 115 pale red colonies

CIX-SUC plate: 501 small blue colonies, 1 pale red colony

This suggests a good degree of counter-selection. Further experiments are in progress.

Plates with 10% sucrose (right) or without (left). There are two control lines with lacZ+ (down) and two test lines Plac-RFP-sacB (up) on each plate. One of the test lines has grown much more poorly with sucrose, the other has failed to grow at all. The inconsistent pink colour suggests a high rate of plasmid loss.

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