Difference between revisions of "Part:BBa K562007:Experience"

Revision as of 12:01, 1 October 2011

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Applications of BBa_K562007

User Reviews

UNIQe701f68e6263b78e-partinfo-00000000-QINU

•••

iGEM Dundee 2011

This part was seen work in practice. Synthesis of the four 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_K562007 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_K562007. Radiolabelled revealed bands of the corresponding sizes to the PduA, PduT and PduU proteins. PduB can be expressed as a smaller PduB' variant from an alternative translation start site. The RED ARROW points to the lane producing polypeptides from BBa_K562007.

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-This part was seen work in practice. Synthesis of the seven encoded polypeptides in very small scale cultures (1 ml) was observed by 35-S-Methionine labelling (Figure 1).
+This part was seen work in practice. Synthesis of the four encoded polypeptides in very small scale cultures (1 ml) was observed by 35-S-Methionine labelling (Figure 1).
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 ===Results===
 <i>E. coli</i> was transformed with <partinfo>BBa_K562007</partinfo> 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 <i>E. coli</i> 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.