Difference between revisions of "Part:BBa K562008:Experience"

Latest revision as of 12:37, 1 October 2011

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Applications of BBa_K562008

User Reviews

UNIQ3dc4208eae3fe679-partinfo-00000000-QINU

•••

iGEM Dundee 2011

This part was seen work in practice. Synthesis of the five 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_K562008 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. Protein labelling was analysed by SDS-PAGE and autoradiography.

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_K562008. Radiolabelled revealed bands of the corresponding sizes to the PduA, PduN, 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_K562008.

UNIQ3dc4208eae3fe679-partinfo-00000004-QINU

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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 five encoded polypeptides in very small scale cultures (1 ml) was observed by 35-S-Methionine labelling (Figure 1).
 |}
 ;
 ===Results===
-<i>E. coli</i> was transformed with <partinfo>BBa_K562008</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.
+<i>E. coli</i> was transformed with <partinfo>BBa_K562008</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.  Protein labelling was analysed by SDS-PAGE and autoradiography.
 This very small scale and sensitive technique reveals all polypeptides being expressed from the plasmid.