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]''.
-Ziel der Gruppe Transport ist es einige für den Transport von Terephthalsäure (TPA) verantwortlichen Proteine aus Comamonas testosteroni KF-1 in Escherichia coli zu exprimieren. TPA soll von E.coli aufgenommen werden, um es für den Metabolismus zugänglich zu machen und anschließend in andere für uns nützliche Stoffe umzuwandeln.
+[[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.]]
-Terephthalsäure kann nur bei niedrigtem pH-Wert die Membran passieren, dies beeinflusst jedoch das Wachstum von E.coli sehr negativ. Daher versuchen wir ein Transportsystem zu finden und zu charakterisieren, das den Membrantransport unter optimalen Wachstumsbedingungen für E.coli ermöglicht.
+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.
-Bei den aus C.testosteroni stammenden Proteinen handelt sich laut Literaturangaben um ein Tripartite Tricarboxylat Transportersystem, welches aus den drei Untereinheiten A, B und C besteht. A ist ein größeres Transmembranprotein mit 12 Transmembrandomänen, B ein kleineres mit nur 4 Transmembrandomänen. C ist ein spezifisches periplasmatisches Bideprotein, das sich frei im Periplasma bewegt und an AB binden kann. Von der Funktion her ähneln sie den ABC-Transportern, jedoch besteht keine Ähnlichkeit in der Sequenz. C.testosteroni KF-1 besitzt zwei unterschiedliche A und B Proteine (A1 mit 505 aa, B1 mit 197 aa und A2 mit 503 aa, B2 mit 162 aa), die unspezifisch für viele verschiedene Substrate sind. A1B1C und A2B2C sollen zunächst auf den Plasmiden pSB1C3 und pSB1A2 in E.coli DH5α eingebracht werden und danach in einen Überexpressionsstamm, z.B. BL21(DE3)pLysS oder c 43 (de3) überfüht werden und unter Regulation eines Arabinosepromotors exprimiert werden.
+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.
-:[[File:Ttt.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]. -->
-Da das Transportsystem bislang noch nicht ausreichend charakterisiert wurde, wird zunächst geprüft welche Komponenten essentiell für den Transportmechanismus sind, bzw. welche Kombination der Untereinheiten überhaupt den Transport von TPA ins Zellinnnere ermöglicht.
+<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>
-Die Aufnahmeraten von TPA werden massenspektrometrisch und photometrisch überprüft. Mit anschließenden Funktionstests wird das Transprotsystem weiter charakterisiert.
-continue with [https://2012.igem.org/Team:TU_Darmstadt/Project/Metabolism 3. Metabolism]