Difference between revisions of "Team:BABS UNSW Australia/project/phlow"
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<figure><center><img src="https://static.igem.org/mediawiki/2015/a/ac/Unsw2015_laczassay_ph_column_graph.jpeg" style="PADDING-BOTTOM:20px;PADDING-TOP:20px;"></center></figure> | <figure><center><img src="https://static.igem.org/mediawiki/2015/a/ac/Unsw2015_laczassay_ph_column_graph.jpeg" style="PADDING-BOTTOM:20px;PADDING-TOP:20px;"></center></figure> | ||
| − | <p>As can be seen here, there is a slight increase in promoter activity at lower pH’s for both ASR and p170. For the purposes of the pHlow plasmid we ideally desire a promoter that will have minimal expression under normal conditions, as can be seen below the overall activity of either promoter is significantly less than CP44 | + | <p>As can be seen here, there is a slight increase in promoter activity at lower pH’s for both ASR and p170. For the purposes of the pHlow plasmid we ideally desire a promoter that will have minimal expression under normal conditions, as can be seen below the overall activity of either promoter is significantly less than CP44. Further characterization attempts should look at using a weaker promoter for the standard comparison. |
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| − | < | + | <figure><center><img src="https://2015.igem.org/File:Unsw2015_laczassay_ph_odnormalised_graph.jpeg" style="PADDING-BOTTOM:20px;PADDING-TOP:20px;"></center></figure> |
<p>Further characterization attempts should look at using a weaker promoter for the standard comparison.</p> | <p>Further characterization attempts should look at using a weaker promoter for the standard comparison.</p> | ||
<p> While there is evidence that both ASR and p170 can be used to upregulate expression under acidic conditions, they still have expression in a neutral environment. While this may be helpful for future iGEM projects the pHlow plasmid relies on a tightly regulated and timed expression of listeriolysin and cre-recombinase, something that these two promoters don’t appear to confer. </p> | <p> While there is evidence that both ASR and p170 can be used to upregulate expression under acidic conditions, they still have expression in a neutral environment. While this may be helpful for future iGEM projects the pHlow plasmid relies on a tightly regulated and timed expression of listeriolysin and cre-recombinase, something that these two promoters don’t appear to confer. </p> | ||
Revision as of 14:08, 18 September 2015
Overview
The two proteins essential for bacterial invasion of mammalian cytoplasm, invasin and listeriolysin, were already in the registry, and were previously shown to work in a very simple genetic circuit - promoter-rbs-protein-rbs-protein. Our aim with the ‘pHlow’ system was to more finely tune the expression of these proteins, as well as improve the biosafety of modified organisms.
Acid shock response promoter
An essential component of the pHlow plasmid is listeriolysin only being expressed once the cell in within the lysosome. Bacteria often are subjected to environmental stressors in the form of pH change, requiring differential gene expression, and hence have pH specific promoters. We have had these endogenous promoters synthesized and have characterised them for use in the pHlow plasmid.
E. Coli ASR Promoter
The acid shock response protein is present in the genome of E. Coli K12 and was first submitted into the registry by IIT Delhi 2013 (http://parts.igem.org/Part:BBa_K1170000) . This part was not available from the registry so we had it synthesised as a gBLOCK from IDT and biobricked it so that future years can access the part without synthesis.
Lactococcus Promoters
Lactococcus lactis produce lactate in their own environment and have several genes that are upregulated due to the low pH they create.The two promoters we are investigating are p170 (BBa_K1677300) and p170-CP25 (BBa_K1677301).The reasons for attempting to characterise both promoters is due to their relative levels of expression; p170 has a higher level of expression, but is a more leaky promoter, whereas p170-CP25 is more tightly regulated. P170 has been characterised to be induced at pH 6.5 and pH 6.0 but only when the culture is in stationary phase [1]. It is important to note that all characterization of these promoters occurred in E. Coli, not L. lactis and that further characterisation needs to be undertaken to conclusively determine their results.
As can be seen here, there is a slight increase in promoter activity at lower pH’s for both ASR and p170. For the purposes of the pHlow plasmid we ideally desire a promoter that will have minimal expression under normal conditions, as can be seen below the overall activity of either promoter is significantly less than CP44. Further characterization attempts should look at using a weaker promoter for the standard comparison.
Further characterization attempts should look at using a weaker promoter for the standard comparison.
While there is evidence that both ASR and p170 can be used to upregulate expression under acidic conditions, they still have expression in a neutral environment. While this may be helpful for future iGEM projects the pHlow plasmid relies on a tightly regulated and timed expression of listeriolysin and cre-recombinase, something that these two promoters don’t appear to confer.
References
[1] Madsen, S. M., Arnau, J., Vrang, A., Givskov, M., & Israelsen, H. (1999). Molecular characterization of the pH‐inducible and growth phase‐dependent promoter P170 of Lactococcus lactis. Molecular microbiology, 32(1), 75-87