Difference between revisions of "Part:BBa K1669001"

Revision as of 21:50, 17 September 2015

CtfA + His-tag

This part includes the CtfA gene from Clostridium acetobutylicum (NCBI: CAP0163) with a C-terminal His-tag, which has been codon optimized for expression in E. coli. This is one of the two polipeptide chains forming the CtfAB (CoA-transferase) enzyme, that is in Clostridium acetobutylicum responsible for conversion of butyrate to butyryl-CoA.

The sequence contains a C-terminal His-tag for purification of the protein using the immobilized-metal affinity chromatography and detected with the anti-His antibodyes.

Usage and Biology

This BioBrick was ligated as a forward insert into a vector with a double terminator (BBa_K823017). The fused construct was then ligated as a back insert into a vector with a strong promoter and a strong RBS (BBa_K608002). We gained positive results trough colony PCR.

To investigate the activity of the construct we utilized SDS-PAGE and Western blot. Escherichia coli DH5α bacteria were transformed with the expression plasmids and grown in shaker flasks at 37 °C in LBC medium overnight. 1 ml samples were taken for analysis. Cells were collected by centrifugation at 6000g for 10 min and resuspended in 100 µl of H20. 10 µl samples were taken and 5 µl of SDS-PAGE loading buffer was added. Samples were cooked at 100 °C for 10 min and resolved on 12% SDS-PAGE gel.

Proteins were detected using Western blot as well. 12% SDS-PAGE gels were electroblotted for 1, 5 hours at 200 V, blocked in non-fat dry milk overnight and incubated the next day in two steps. A dilute solution of primary antibody was incubated with the membrane under gentle agitation for an hour. Then a dilute solution was incubated with the membrane under gentle agitation for an hour as well. The hydrogen peroxide method was for visualization.

Sequence and Features