Team:TU Darmstadt/Project/Transport

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Latest revision as of 01:00, 27 September 2012

Transport

The objective of group "Transport" is the integration of a terephtalic acid uptake system in Escherichia coli (E. coli). The uptake of TPA is crucial to produce high-value molecules into our host bacteria. TPA can only pass the membrane at low pH values, which adversely affects the growth of [http://en.wikipedia.org/wiki/E._coli E. coli]. Therefore, a suitable transport system is needed that operates under optimal growth conditions for [http://en.wikipedia.org/wiki/E._coli E. coli].

Figure 1. The mechanism of terephtalalic acid (TPA ) uptake: Protein C binds the TPA and transfers it to the Proteins A and B, which transport the TPA across the inner membrane.

According to the data published by Sasoh et al.^[1] Comamonas testosteroni (C. testosteroni) is able to utilize TPA as the sole carbon and energy source. For that reason, we decided to isolate the putative TPA uptake System of C. testosteroni. This system belongs to the tripartite tricarboxylate transporters and consists of three subunits (A to C). The large subunit A is a with 11-12 alpha-helical transmembrane protein. It is acompanied by the small transmembrane subunit B which constis of 4-5 alpha-helical transmembrane protein. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 Subunit C] is a specific periplasmic binding protein, which is moving freely in the periplasmic space and bonds to the AB units. (Fig.1) The function is similar to ABC transporters, however the sequences are unrelated. C. testosteroni features two different configurations of A and B proteins ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 A1] with 505 amino acids (aa) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 B1] with 197 aa or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 A2] with 503 aa and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 B2] with 162 aa). The proteins are naturally unspecific and can transport different substrates.

We designed under the control of a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Arabinose inducible promotor](AraC-Pbad) two putative terephtalate uptake operons. The intake of TPA was checked by photometry,gas chromatography-mass spectrometry (GC-MS) and energy dispersive X-ray spectroscopy (EDX). To determine the essential components and their combination for TPA transport into the cell, the genes were expressed in an overexpression strain E. coli C43(DE3). The structure characterisation was done by some bioinformatical tools like Protein Homology/anologY Recognition Engine V 2.0 (PHYRE2), I-TASSER servers, SignalIP 4.0 Server and TatP 1.0 Server. Additionaly, we developed a new method (ATS-method) to detect TPA with GC-MS.Futhermore, we characterized the AraC-Pbad regulation unit.

Detailed information on our approach is available in the Transport Labjournal. The next section contains information on the Metabolism step.

[1] Sasoh, M., E. Masai, et al. (2006). "Characterization of the terephthalate degradation genes of Comamonas sp. strain E6." Appl Environ Microbiol 72(3): 1825-1832.

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 == Transport ==
+<!-- [[File:Transport_project.png|150px|right]] -->
+The objective of group "[https://2012.igem.org/Team:TU_Darmstadt/Team#Transport Transport]" is the integration of a [https://2012.igem.org/Team:TU_Darmstadt/Materials/TPA terephtalic acid] uptake system in ''Escherichia coli''  (''E. coli''). The uptake of [https://2012.igem.org/Team:TU_Darmstadt/Materials/TPA TPA] is crucial to produce high-value molecules into our host bacteria. [https://2012.igem.org/Team:TU_Darmstadt/Materials/TPA TPA] can only pass the membrane at low pH values, which adversely affects the growth of ''[http://en.wikipedia.org/wiki/E._coli E. coli]''. Therefore, a suitable transport system is needed that operates under optimal growth conditions for [http://en.wikipedia.org/wiki/E._coli E. coli]''.
-The goal for group "Transport" is to express ''comamonas testosteroni'' KF-1 transport proteins in ''Escherichia coli''. The target proteins are regulating the Terephthalic acid (TPA) transport. The TPA transport is crucial to make it accessible for metabolism to useful substances in ''E. coli''.
+[[File:Ttt_v5.png|450px|thumb|right|Figure 1. '''The mechanism of [https://2012.igem.org/Team:TU_Darmstadt/Materials/TPA terephtalalic acid] ([https://2012.igem.org/Team:TU_Darmstadt/Materials/TPA TPA] ) uptake:''' Protein C binds the [https://2012.igem.org/Team:TU_Darmstadt/Materials/TPA TPA] and transfers it to the Proteins A and B, which transport the [https://2012.igem.org/Team:TU_Darmstadt/Materials/TPA TPA] across the inner membrane.]]
-TPA can only pass the membrane at low pH values, which negatively affect the growth of ''E. coli''. Therefore, a suitable transport system is needed that operates under good growth conditions for ''E. coli''.
+According to the data published by ''Sasoh et al.''<sup>[1]</sup> ''Comamonas testosteroni'' (''C. testosteroni'') is able to utilize [https://2012.igem.org/Team:TU_Darmstadt/Materials/TPA TPA] as the sole carbon and energy source. For that reason, we decided to isolate the putative TPA uptake System of ''C. testosteroni''. This system belongs to the tripartite tricarboxylate transporters and consists of three subunits (A to C). The large subunit A is a with 11-12 alpha-helical transmembrane protein. It is acompanied by the small transmembrane subunit B which constis of 4-5 alpha-helical transmembrane protein. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808001 Subunit C] is a specific periplasmic binding protein, which is moving freely in the periplasmic space and bonds to the AB units. (Fig.1) The function is similar to ABC transporters, however the sequences are unrelated. ''C. testosteroni'' features two different configurations of A and B proteins ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K808002 A1] with 505 amino acids (aa) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808003 B1] with 197 aa or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808004 A2] with 503 aa and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808005 B2] with 162 aa). The proteins are naturally unspecific and can transport different substrates.
-Based on literature the proteins from ''C.testosteroni'' are forming a tripartite tricarboxylate transport system. The system consists of three subunits called A, B and C. A is a large transmembrane protein with 12 transmembrane domains. It is acompanied by a smaller transmembrane protein with only 4 transmembrane domains. C is a specific periplasmatic bonding protein, which is moving freely in the periplasm and bonds to the AB units. The function is similar to ABC transporters, however the sequences are unrelated. ''C.testosteroni'' KF-1 owns two different A and B proteins (A1 with 505 aa, B1 with 197 aa and A2 with 503 aa, B2 with 162 aa). The proteins are naturally unspecific and can transport different substrates. Initially A1B1C and A2B2C shall be insert on pSB1C3 and pSB1A2 plasmides in ''E. coli'' DH5α. Afterwards they will be transferred in an overexpression strain like BL21(DE3)pLysS or c43 (de3) and expressed under arabinose promotor regulation.
+We designed under the control of a [http://partsregistry.org/wiki/index.php?title=Part:BBa_K808000 Arabinose inducible promotor](AraC-Pbad) two putative terephtalate uptake operons. The intake of [https://2012.igem.org/Team:TU_Darmstadt/Materials/TPA TPA] was checked by photometry,gas chromatography-mass spectrometry (GC-MS) and energy dispersive X-ray spectroscopy ([https://2012.igem.org/Team:TU_Darmstadt/Protocols/TEM-EDX#TEM-EDX-Measurement EDX)]. To determine the essential components and their combination for [https://2012.igem.org/Team:TU_Darmstadt/Materials/TPA TPA] transport into the cell, the genes were expressed in an overexpression strain ''E. coli C43(DE3)''. The structure  characterisation was done by some bioinformatical tools like '''P'''rotein '''H'''omology/anolog'''Y''' '''R'''ecognition '''E'''ngine V 2.0 (PHYRE2), I-TASSER  servers, SignalIP 4.0 Server and TatP 1.0 Server. Additionaly, we developed a new method ([https://2012.igem.org/Team:TU_Darmstadt/Protocols/GC-MS ATS-method)] to detect TPA with [https://2012.igem.org/Team:TU_Darmstadt/Protocols/GC-MS GC-MS].Futhermore, we characterized the AraC-Pbad regulation unit.
-:https://static.igem.org/mediawiki/igem.org/f/f8/Ttt_v3.png
+Detailed information on our approach is available in the [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Transport Transport Labjournal]. The next section contains information on the [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism Metabolism] step.
+<!--For more informations concerning the other projects continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism 3. Metabolism]. -->
+<span style="font-size:9px;">[1] Sasoh, M., E. Masai, et al. (2006). "Characterization of the terephthalate degradation genes of Comamonas sp. strain E6." Appl Environ Microbiol 72(3): 1825-1832.</span>
-Insufficient investigation of the transport system became necessary to find the essential components and their combination for TPA transport into the cell.
-The intake of TPA is checked by mass spectrometry and photometry. Subsequent functional tests are further characterizing the transport system.
-continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism 3. Metabolism]