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

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
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===User Reviews===
 
===User Reviews===
 
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<partinfo>BBa_K562006 AddReview number</partinfo>
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<partinfo>BBa_K190019 AddReview 3</partinfo>
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<I>Username</I>
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<I>iGEM Dundee 2011</I>
 
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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).
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===Results===
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<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.  Protein labelling was analysed by SDS-PAGE and autoradiography.
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This very small scale and sensitive technique reveals all polypeptides being expressed from the plasmid.
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[[Image:2006.jpg]]
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: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.
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Latest revision as of 12:34, 1 October 2011

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.

Applications of BBa_K562006

User Reviews

UNIQ24e2782bd9a208df-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. Protein labelling was analysed by SDS-PAGE and autoradiography.

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

2006.jpg

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.



UNIQ24e2782bd9a208df-partinfo-00000004-QINU