Difference between revisions of "Part:BBa K339008"
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===Functional Parameters=== | ===Functional Parameters=== | ||
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'''Characterization of the degP promoter's response to folding and misfolding proteins''' | '''Characterization of the degP promoter's response to folding and misfolding proteins''' | ||
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<p> Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pDegP coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then subcultured in 20 ml LB broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose. This was incubated in the shaker for 12-14 hours and RFP output was measured using 555/632 nm.</p> | <p> Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pDegP coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then subcultured in 20 ml LB broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose. This was incubated in the shaker for 12-14 hours and RFP output was measured using 555/632 nm.</p> | ||
| − | + | "http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/DegPinduction.png" | |
<p> This figure demonstrates that the DegP promoter activated with 15 different concentrations of arabinose. This diagram shows that the DegP promoter (K239000) is not particularly sensitive to misfolding proteins.</p> | <p> This figure demonstrates that the DegP promoter activated with 15 different concentrations of arabinose. This diagram shows that the DegP promoter (K239000) is not particularly sensitive to misfolding proteins.</p> | ||
Latest revision as of 04:32, 30 October 2010
degP Promoter With RFP Reporter
degP promoter upstream of an RFP construct (BBa_I13507).
Usage and Biology
This construct was used in order to characterize the degP promoter, specifically its response to misfolded and folded proteins in the periplasm of Escherichia Coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 814
Illegal AgeI site found at 926 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
Characterization of the degP promoter's response to folding and misfolding proteins
Protocol
Arabinose inducible promoter (I0500) coupled with standard ribosome binding site (B0034) and the respective maltose binding protein were transformed into competent cells containing pDegP coupled with RFP generator (I13507). These cells were plated and incubated overnight. Colonies from each of the plates were selected and overnight cultures were prepared at 37 C. These 5 ml overnight cultures were then subcultured in 20 ml LB broth. These were shaken for 6-8 hours and aliquoted into 5 ml cultures and induced with varying levels of arabinose. This was incubated in the shaker for 12-14 hours and RFP output was measured using 555/632 nm.
"http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/DegPinduction.png"
This figure demonstrates that the DegP promoter activated with 15 different concentrations of arabinose. This diagram shows that the DegP promoter (K239000) is not particularly sensitive to misfolding proteins.
Discussion and conclusions
The figure shows that MalE and MalE31 express the DegP promoter in a similar fashion. This is slightly contradictory compared to the literature. The literature claims that the DegP promoter is upregulated in the case of protein misfolding, graph shown . Since MalE and MalE31 have been tested using other experiments described in this page, it is reasonable to conclude that K230009 is not very responsive to protein folding stress, that is , the DNA might not be consistent.