Part:BBa_K917010:Experience

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Applications of BBa_K917010

This part is designed for use in selection-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. The kanamycin resistance determinant, BBa_P1003, has been added so that both selection and counter-selection are possible. Growth occurred on plates containing chloramphenicol (40 mg/l) and kanamycin (50 mg/l) indicating correct function of P1003 (data not shown).

An quick preliminary trial was conducted. 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.

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