Team:Imperial College/CAT


Chloramphenicol Acetyltransferase Characterisation

We have characterised the chloramphenicol concentrations at which chlorampehnicol acetyltransferase (Part: BBa_J31005) (CAT) will protect cells, allowing them to grow and thrive. This is essential as it allows us to make the correct selections when using the CAT gene.

If a culture of cells becomes contaminated with another chloarmphenicol resistant bacterium and we lack an extra resistance marker, the upper limit at which our cells can survive in chloramphenicol may allow the removal of the contaminating strain or species.

To this end, characterisation of chloramphenicol acetyltransferase was carried out according to this protocol.

To characterise chloramphenicol acetyltransferase, a construct containing the CAT gene, expression equipment, a fluorescent protein gene (GFP or RFP) and the 5' and 3' amyE integration sequences was assembled and integrated into B. subtilis. This modified B. subtilis was then used to characterise CAT.

CAT Results.PNG

CAT Working Concentration.PNG

The construct containing the CAT gene was integrated into the B. subtilis genome for characterisation. As such, the CAT gene is single copy only. CAT will be able to protect a cell from higher levels of chloramphenicol if it is present in a construct on a plasmid (particularly a high copy number plasmid).

Chloramphenicol can be used for selection up to a concentration of 30 μg/ml. If a higher concentration is required, a bacterium possessing a single highly expressed copy of chloramphenicol acetyltransferase can survive (with difficulty) at up to 75 μg/ml of chloramphenicol.