Difference between revisions of "Part:BBa K562005:Experience"
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===Results=== | ===Results=== | ||
| − | <i>E. coli</i> was transformed with <partinfo>BBa_K562005</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_K562005</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. | This very small scale and sensitive technique reveals all polypeptides being expressed from the plasmid. | ||
Latest revision as of 12:33, 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_K562005
User Reviews
UNIQf0fa0aabe03e0477-partinfo-00000000-QINU
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iGEM Dundee 2011 |
This part was seen work in practice. Synthesis of the single encoded polypeptide in a very small scale culture (1 ml) was observed by 35-S-Methionine labelling (Figure 1). |
Results
E. coli was transformed with BBa_K562005 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_K562005. Radiolabelled revealed a band of the corresponding size to the PduN protein. The RED ARROW points to the lane producing polypeptides from BBa_K562005.
UNIQf0fa0aabe03e0477-partinfo-00000004-QINU