Difference between revisions of "Part:BBa K1149003"
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To ensure secretion in E.coli, we have also added a [https://parts.igem.org/Part:BBa_K1149023 pelB] seretion tag to the N-terminal. We also added a 6xHistidine tag for Western blotting and protein purification purposes. | To ensure secretion in E.coli, we have also added a [https://parts.igem.org/Part:BBa_K1149023 pelB] seretion tag to the N-terminal. We also added a 6xHistidine tag for Western blotting and protein purification purposes. | ||
| − | + | PUR degradation by esterase: | |
| − | https://static.igem.org/mediawiki/ | + | https://static.igem.org/mediawiki/parts/3/31/PUR_esterase_theoretical_pathway_2.jpg |
| + | <h2>Characterisation</h2> | ||
| + | Our PueA construct was tested for growth upon induction by addition of Arabinose. | ||
| − | + | https://static.igem.org/mediawiki/2013/thumb/e/e6/PueA.png/450px-PueA.png | |
| − | + | ||
| − | + | '''PueA growth assay.''' MG1655'' E. coli'' were grown for 6h with 0 μM or 6 μM Arabinose. There was no difference between no induction and induction as a t-test gave p = 0.5559 > 0.05. Growth was at 37°C in LB with shaking. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM. | |
| − | Stern, R. V., and G. T. Howard. 2000. The polyester polyurethane gene (pueA) from Pseudomonas clororaphis encodes lipase. FEMS Microbiol. Lett. 185:163–168. | + | |
| + | <b>Conclusion: There is no growth inhibition caused by induction in PueA. </b> | ||
| + | |||
| + | <h2>References: </h2> | ||
| + | |||
| + | <p>[http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2010.00231.x/full RTX proteins: a highly diverse family secreted by a common mechanism ]</p> | ||
| + | <p>Stern, R. V., and G. T. Howard. 2000. The polyester polyurethane gene (pueA) from Pseudomonas clororaphis encodes lipase. FEMS Microbiol. Lett. 185:163–168.</p> | ||
Latest revision as of 23:46, 2 October 2013
pelB-pueA expression
This part expresses a plastic degradation enzyme under the control of xylose inducible promoter.
The enzyme is the pueA from Pseudomonas chlororaphis, that encodes carboxylesterase that breaks down ester bonds in polyurethane. This protein is [http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2010.00231.x/full naturally extracellular], it has a C-terminal secretion signal located downstream of a domain containing three glycine-rich repeats. These glycine-rich repeats are nonapeptide motifs involved in Ca2+-binding found in proteins secreted via type I systems. It uses the RTX system. To ensure secretion in E.coli, we have also added a pelB seretion tag to the N-terminal. We also added a 6xHistidine tag for Western blotting and protein purification purposes.
PUR degradation by esterase:
Characterisation
Our PueA construct was tested for growth upon induction by addition of Arabinose.
PueA growth assay. MG1655 E. coli were grown for 6h with 0 μM or 6 μM Arabinose. There was no difference between no induction and induction as a t-test gave p = 0.5559 > 0.05. Growth was at 37°C in LB with shaking. Error bars are SEM, n=4. Figure made by Imperial College London 2013 iGEM.
Conclusion: There is no growth inhibition caused by induction in PueA.
References:
[http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6976.2010.00231.x/full RTX proteins: a highly diverse family secreted by a common mechanism ]
Stern, R. V., and G. T. Howard. 2000. The polyester polyurethane gene (pueA) from Pseudomonas clororaphis encodes lipase. FEMS Microbiol. Lett. 185:163–168.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 99
Illegal BglII site found at 964 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]