Team:TU Darmstadt/Project/Transport

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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 a E. coli. TPA can only pass the membrane at low pH values, which adversely affects the growth of E. coli. Therefore, a suitable transport system is needed that operates under optimal growth conditions for E. coli. According to the data published by Sasoh et al.^[1] Comamonas testosteroni (C. testosteroni) is able to utilizes TPA as the sole carbon and energy source. For that reason, we decided to isolate the putative TPA uptake System from C.testosteroni . This System bolongs to the tripartite tricarboxylate transporters and consists of three subunits (A to C). The large subunit A is a transmembrane protein with 11-12 alpha-helical spanners. It is acompanied by the small transmembrane subunit B which constis of 4-5 alpha-helical spanners. 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 owns two different A and B proteins (A1 with 505 amino acids (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 was isolated from C. testosteroni and be insert on pSB1C3 and pSB1A2 plasmides in E. coli DH5α. The Genes are regulated under the control of a Arabinose inducible promotor (araC-Pbad ). The intake of TPA is 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 like 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.

Figure 1. The mechanism of terephtalalic acid (TPA) uptake. Protein C is binding the TPA and brings it to the Proteins A and B, which transports the TPA across the inner membrane.

Detailed information on our approach is available in the Transport Labjournal. For more informations concerning the other projects continue with 3. Metabolism.

[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]]
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-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''.
+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 a E. coli. TPA can only pass the membrane at low pH values, which adversely affects the growth of E. coli. Therefore, a suitable transport system is needed that operates under optimal  growth conditions for E. coli.
+According to the data published by Sasoh et al.<sup>[1]</sup> Comamonas testosteroni (C. testosteroni) is able to utilizes TPA as the sole carbon and energy source. For that reason, we decided to isolate the putative TPA uptake System from C.testosteroni . This System bolongs to the tripartite tricarboxylate transporters and consists of three subunits (A to C). The large subunit A is a transmembrane protein with 11-12 alpha-helical spanners. It is acompanied by the small transmembrane subunit B which constis of  4-5 alpha-helical spanners. 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 owns two different A and B proteins (A1 with 505 amino acids (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 was isolated from C. testosteroni  and be insert on pSB1C3 and pSB1A2 plasmides in ''E. coli  DH5&alpha;''. The Genes are regulated under the control of a Arabinose inducible promotor (araC-Pbad ).
-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''.
+The intake of TPA is 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 like 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.
-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.
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-Due to insufficient investigation of the transport system it became necessary for us to determine the essential components and their combination for TPA transport into the cell.
+Figure 1. '''The mechanism of terephtalalic acid (TPA) uptake.''' Protein C is binding the TPA and brings it to the Proteins A and B, which transports the TPA across the inner membrane.
-The intake of TPA is checked by mass spectrometry and photometry. Subsequent functional tests are further characterizing the transport system.
 Detailed information on our approach is available in the [https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Transport Transport Labjournal]. For more informations concerning the other projects continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism 3. Metabolism].
+[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.