Difference between revisions of "Part:BBa K562006:Experience"

Revision as of 11:59, 1 October 2011

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Applications of BBa_K562006

User Reviews

UNIQ054277bc82acf297-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.

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+===Results===
+<i>E. coli</i> was transformed with <partinfo>BBa_K562006</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.
+This very small scale and sensitive technique reveals all polypeptides being expressed from the plasmid.
+[[Image:2006.jpg]]
+:Figure 1: Transcription and translation of gene products produced from <partinfo>BBa_K562006</partinfo>. 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.
 <!-- DON'T DELETE --><partinfo>BBa_K562006 EndReviews</partinfo>