Difference between revisions of "Part:BBa K808030"
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====Composition==== | ====Composition==== | ||
− | Our fusion protein BBa_K808030 is highly chimeric. It consists of parts from three different bacteria, which are the following (starting from N-terminus to C-terminus): | + | Our fusion protein BBa_K808030 (Figure 1) is highly chimeric. It consists of parts from three different bacteria, which are the following (starting from N-terminus to C-terminus): |
* [https://parts.igem.org/wiki/index.php?title=Part:BBa_K808028 PhoA] (BBa_K808028) is responsible for the peri plasmatic expression of our chimeric protein. This is highly important because once expressed into peri plasma, our construct will self-install into the outer membrane. PhoA is native to ''E.coli'' | * [https://parts.igem.org/wiki/index.php?title=Part:BBa_K808028 PhoA] (BBa_K808028) is responsible for the peri plasmatic expression of our chimeric protein. This is highly important because once expressed into peri plasma, our construct will self-install into the outer membrane. PhoA is native to ''E.coli'' | ||
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− | [[Image:bba_k808032_schema.png| | + | [[Image:bba_k808032_schema.png|600px|center|thumb|Figure 1: schematic graphic of BBa_K808030, integrated into an biological membrane. The C-terminal[https://parts.igem.org/wiki/index.php?title=Part:BBa_K808027 EstA] (BBa_K808027) consists of a C-terminal ß-Barrel froming the membrane anchor, and a N-terminal inactive esterase domain, carrying the passenger enzyme. In this case the passenger Enzyme is [https://parts.igem.org/wiki/index.php?title=Part:BBa_K808026 pNB-Esterase 13] (BBa_K808026).]] |
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+ | Once this Biobrick is expressed it will develope hydrolytic activities due to its catalytical domain, the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K808026 pNB-Esterase 13] (BBa_K808026). Our characerisation of this chimeric protein was performed with the arabinose inducible operon [https://parts.igem.org/wiki/index.php?title=Part:BBa_K808032 BBa_K808032] | ||
====Visualization==== | ====Visualization==== | ||
− | With special regards to our [http://2012.igem.org/Team:TU_Darmstadt/Project/Simulation Simulation lab] we show you the result of great computional work. Our dry lab made some homologies and edited these beautiful graphics of our chimeric protein. (Figure | + | With special regards to our [http://2012.igem.org/Team:TU_Darmstadt/Project/Simulation Simulation lab] we show you the result of great computional work. Our dry lab made some homologies and edited these beautiful graphics of our chimeric protein. (Figure 2 & 3) |
− | [[Image:pnb_EstA_ohne_mem.png|500px|center|thumb|Figure | + | [[Image:pnb_EstA_ohne_mem.png|500px|center|thumb|Figure 2: The composition and folding of BBa_K808030. Yellow:[https://parts.igem.org/wiki/index.php?title=Part:BBa_K808026 pNB-Esterase 13] (BBa_K808026)after the N-terminal cut-off of PhoA. Gray: inactive catalytical part of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K808027 EstA] (BBa_K808027). Blue: Transmembrane helix of EstA, connecting its inactive catalytical domain and membrane anchor. Red: Anchor domain of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K808027 EstA] (BBa_K808027)]] |
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− | [[Image:pnb_EstA.png|500px|center|thumb|Figure | + | [[Image:pnb_EstA.png|500px|center|thumb|Figure 3: The composition and folding of BBa_K808030 when integrated into a biological membrane]] |
Latest revision as of 21:15, 25 September 2012
RBS-PhoA-His6tag-pNBEst13-Myctag-EstA
RBS-PhoA-His6tag-pNBEst13-Myctag-EstA is a long name for a long protein construct. It is a fusion protein which is secreted into the peri plasma in order to be integrated into the outer membran. When the phoA signal sequence is cut off, the N-terminus is formed by our pNB-Est13. This esterase shows actitivty towards PET and is anchored onto the cell by its inactive EstA membrane anchor.
Annotation: This site is about the construct and composition of our chimeric protein BBa_K808030. For any information about its expression rate or enzyme activity please visit BBa_K808032
Usage and Biology
Composition
Our fusion protein BBa_K808030 (Figure 1) is highly chimeric. It consists of parts from three different bacteria, which are the following (starting from N-terminus to C-terminus):
- PhoA (BBa_K808028) is responsible for the peri plasmatic expression of our chimeric protein. This is highly important because once expressed into peri plasma, our construct will self-install into the outer membrane. PhoA is native to E.coli
- pNB-Esterase 13 (BBa_K808026) is the the catalytical domain of BBa_K808030. It derives from an esterase of Bacillus licheniformis.
- EstA (BBa_K808027) serves as a membrane anchor but is an inactive and membrane bound esterase mutant deriving from Pseudomonas aeruginosa
![](/wiki/images/b/bf/Bba_k808032_schema.png)
Once this Biobrick is expressed it will develope hydrolytic activities due to its catalytical domain, the pNB-Esterase 13 (BBa_K808026). Our characerisation of this chimeric protein was performed with the arabinose inducible operon BBa_K808032
Visualization
With special regards to our [http://2012.igem.org/Team:TU_Darmstadt/Project/Simulation Simulation lab] we show you the result of great computional work. Our dry lab made some homologies and edited these beautiful graphics of our chimeric protein. (Figure 2 & 3)
![](/wiki/images/4/45/Pnb_EstA_ohne_mem.png)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1174
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1700
Illegal NgoMIV site found at 1838
Illegal NgoMIV site found at 2378
Illegal NgoMIV site found at 2747
Illegal AgeI site found at 1245 - 1000COMPATIBLE WITH RFC[1000]