Part:BBa_K562006:Experience

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

User Reviews

UNIQ5721185c93567012-partinfo-00000000-QINU

••• iGEM Dundee 2011

This part was seen work in practice. Synthesis of the two encoded polypeptides in very small scale cultures (1 ml) was observed by 35-S-Methionine labelling (Figure 1).

Results

E. coli was transformed with BBa_K562006 expressed from pT7.5, and the plasmid pGP1-2 encoding T7 polymerase, and grown aerobically for 3 hours in LB medium. T7 polymerase synthesis was induced by heat shock before all native transcription in E. coli was halted by the addition of rifampicin. 10 minutes later 1 ml of cells was then collected and spiked with 35-S-labelled Methionine. Following a 10 minute incubation the cell pellet was harvested and resuspended in Laemmli disaggregation buffer.

This very small scale and sensitive technique reveals all polypeptides being expressed from the plasmid.

Figure 1: Transcription and translation of gene products produced from BBa_K562006. Radiolabelled revealed bands of the corresponding sizes to the PduT and PduU proteins. The RED ARROW points to the lane producing polypeptides from BBa_K562006.

UNIQ5721185c93567012-partinfo-00000004-QINU