Team:Peking S/project/wire

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Chemical Wire Toolbox

Introduction|Harvesting ‘Chemical Wires’ From Nature|Synthesizing Quorum Sensing Inverters|Orthogonal Activating Matrix

Introduction to ‘Chemical Wire’ toolbox

Cell-cell communication-based multicellular networks provide an extended vista for synthetic biology. By compartmentalizing complex genetic circuits into separate engineered cells, the difficulty of the construction by layering elementary gates can be dramatically reduced, partly due to the insulation of crosstalk between modules, the suppression of noise by populationally averaging, and the reducing of metabolic burden in host cells. What’s more, cell-cell communication-based multicellular feature enables coordination and synchronization among cells in and between populations and facilitates the generation of reliable non-Boolean dynamics.

However, orthogonal ‘chemical wires’ that allow concurrent communication are far from sufficient, making developing a versatile ‘chemical wire’ toolbox for conducting a complex gene network more necessary. This year we are aiming to develop a ‘chemical wire’ toolbox, applicable for both Boolean and Non-Boolean gene networks.

As natural quorum sensing systems provide an excellent pool for developing ‘chemical wire’ toolbox, harvesting ‘chemical wires’ from the nature is a fast and affordable way. We selected and evaluated a recently reported quorum sensing system, CinI-CinR system from Rhizobium leguminosarum as a candidate of our toolbox. An artificial QS system, PchAB-NahR system exploiting salicylate as signaling molecule was also built. Both of them were proved to owe promising performance.

For more details, click here

On the other hand, all of the quorum sensing systems currently exploited in synthetic biology exhibit transcriptional activation, which cannot provide negative feed back loops during cell-cell communication, and the conventional inverter, which was implemented by the repressor-operator pairs, has evident defects. We have developed a ‘from ground up’ approach to synthesize direct, fast and reliable signaling inverters for synthetic microbial consortia, harnessing the conditioned binding of quorum sensing regulators to their cognate DNA boxes to control the accessibility of RNA polymerase.

For more details, click here

With candidates harvested from nature or re-designed from the natural counterpart, we started to characterize them, specially focusing on their orthogonality, dose response, time dependence (signaling speed) and their ability to coordinate cell population.

For more details, click here

Reference

[1] Li, B. You, L. Synthetic biology: Division of logic labour. Nature 469, 171–172 (2011).

[2] Marguet, P. Balagadde F. Tan C. You L. Biology by design: reduction and synthesis of cellular components and behaviour. JR Soc Interface 4, 607–623 (2007).

[3] Voigt, C.A. Genetic parts to program bacteria. Curr Opin Biotechnol 17, 548–557 (2006).

[4] Clancy, K, Voigt, C.A. Programming cells: towards an automated 'Genetic Compiler'. Curr Opin Biotechnol 21, 572–581 (2010) .

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@@ Line 15: / Line 15: @@
-===Harvesting ‘Chemical Wires’ From Nature===
+===Introduction to ‘Chemical Wire’ toolbox===
 <br>
-'''Introduction'''
+Cell-cell communication-based multicellular networks provide an extended vista for synthetic biology. By compartmentalizing complex genetic circuits into separate engineered cells, the difficulty of the construction by layering elementary gates can be dramatically reduced, partly due to the insulation of crosstalk between modules, the suppression of noise by populationally averaging, and the reducing of metabolic burden in host cells. What’s more, cell-cell communication-based multicellular feature enables coordination and synchronization among cells in and between populations and facilitates the generation of reliable non-Boolean dynamics.
-Multicellular strategy provides us a visa to constructing a complex gene network. During gene network constructing, as genetic network scaling up, more cells are involved in compartmentalizing genetic network. Natural quorum sensing and small chemical molecule generator and receiver systems provide an excellent pool for developing ‘chemical wire’ toolbox. Unfortunately, naturally existed and well-characterized cell-cell communication systems are far from sufficient. So harvesting ‘chemical wires’ from the nature is a fast and affordable way for the toolbox development.
+However, orthogonal ‘chemical wires’ that allow concurrent communication are far from sufficient, making developing a versatile ‘chemical wire’ toolbox for conducting a complex gene network more necessary. This year we are aiming to develop a ‘chemical wire’ toolbox, applicable for both Boolean and Non-Boolean gene networks.
-Many signaling molecules could serve as candidates of synthetic consortia ‘chemical wire’, but not all of them perform well when applied to microbial chassis. One of the most significant problems is that they are difficult to be synthesized or perceived when chemical molecule generating or receiving mechanism is too complex to engineer. Besides, threshold, response time and coordination of receiver cell performance should also be considered during chemical wire toolbox candidate selection. Because only chemical signaling molecules with low threshold, fast response speed and highly synchronized coordination response deserve to be candidates.
+As natural quorum sensing systems provide an excellent pool for developing ‘chemical wire’ toolbox, harvesting ‘chemical wires’ from the nature is a fast and affordable way. We selected and evaluated a recently reported quorum sensing system, CinI-CinR system from'' Rhizobium leguminosarum'' as a candidate of our toolbox. An artificial QS system, PchAB-NahR system exploiting salicylate as signaling molecule was also built. Both of them were proved to owe promising performance.
-According to the criteria above, in our project two signal generator and receiver systems were selected. One employs auto-inducer N-(3-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone (3OH-C14:1-HSL), as the signal molecule (Figure 1). The other system employs Salicylate (Figure 2). In order to confirm their usability in practical application, we designed experiment including three aspects of the systems, namely dose response, time dependence and coordination.
-=== Cin system ===
-This system was derived from Rhizobium leguminosarum which are Gram-negative soil bacteria living in symbiotic association with legumes by forming nodules. In this system, cinI, pcin, cinR are core elements. CinI is a typical LuxI-type synthase, synthesizing 3OH-C14:1-HSL and cinR is a LuxR-type transcriptional regulator to sense this molecules. Pcin will be turned on by CinR binding together with 3OH-C14:1-HSL.
-Naturally existing and simple mechanism make Cin system a promising candidate.
-<center>[[File:LM1.png]]</center>
-<center>''Figure 1. N-(3-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone chemical structure''</center>
-Experiments were conducted to verify its validness. Firstly we constructed two plasmids as 3OH-C14:1-HSL generator and receiver devices (Figure 2). Then dose response, ,time dependence and coordination experiments were performed.
-<center>[[File:LM2a.png|340px]][[File:LM2b.png|340px]]</center>
-<center>(A)                                                   (B)</center>
 <center>[[File:LB2cbbb.png|680px]]
-(C)
 </center>
-''Figure 2. Architecture of cin system and schematic of generator and receiver system. (A) Autoinducer generator plasmid. (B) Autoinducer receiver system plasmid. This plasmid contains 3 core parts: constitutively expressed cinR, protein CinR binding promoter pcin and CinR activated gfp. (C) Operation of experiment system.''
+''For more details'', [https://2011.igem.org/Team:Peking_S/project/wire/harvest      ''click here'']
-=== Salicylate system ===
-Some microorganisms can degrade salicylate and the mechanism has been studied extensively in Pseudomonas putida. Interestingly, there is a bacterium species named Pseudomonas aeruginosa which can synthesize salicylate. These two bacteria strains provide the possibility of constructing an artificial quorum sensing system.
+On the other hand, all of the quorum sensing systems currently exploited in synthetic biology exhibit transcriptional activation, which cannot provide negative feed back loops during cell-cell communication, and the conventional inverter, which was implemented by the repressor-operator pairs, has evident defects. We have developed a ‘from ground up’ approach to synthesize direct, fast and reliable signaling inverters for synthetic microbial consortia, harnessing the conditioned binding of quorum sensing regulators to their cognate DNA boxes to control the accessibility of RNA polymerase.
+<center>[[File:NN10.png|400px]]</center>
+''For more details'', [https://2011.igem.org/Team:Peking_S/project/wire/inverter      ''click here'']
-<center>[[File:LM3.png]]</center>
-''Figure 3. Chemical structure of salicylate''
+With candidates harvested from nature or re-designed from the natural counterpart, we started to characterize them, specially focusing on their orthogonality, dose response, time dependence (signaling speed) and their ability to coordinate cell population.
+<center>[[File:signalingOrthogonal.png|450px]]</center>
+''For more details'', [https://2011.igem.org/Team:Peking_S/project/wire/matrix     ''click here'']
-==== 1. Salicylate operon has a broad concentration response curve ====
+== Reference ==
-We found that salicylate sensor NahR is able to response to salicylate among a wide concentration range. When induced with high concentration of salicylate, for instance, 10^-3M, the gene of interest regulated by pSal promoter will be dramatically expressed, while NahR could still sense salicylate even as low as 10^-6M.
+[1] Li, B. You, L. Synthetic biology: Division of logic labour. Nature 469, 171–172 (2011).
-==== 2. Salicylate would not cause huge burden on the cell metabolism ====
+[2] Marguet, P. Balagadde F. Tan C. You L. Biology by design: reduction and synthesis of cellular components and behaviour. JR Soc Interface 4, 607–623 (2007).
+[3] Voigt, C.A. Genetic parts to program bacteria. Curr Opin Biotechnol 17, 548–557 (2006).
-As a chemical wire, salicylate has small molar mass and simple chemical structure. The synthesis pathway of salicylate only involves two genes. As a result, metabolic burden will not be a hurdle when using salicylate as artificial quorum sensing molecules.
+[4] Clancy, K, Voigt, C.A. Programming cells: towards an automated 'Genetic Compiler'. Curr Opin Biotechnol 21, 572–581 (2010) .
-==== 3. Salicylate has a unique chemical feature making it more stable ====
-Benzene ring and carboxyl make salicylate more stable than other quorum-sensing small molecules.
-In order to characterize the system, generator device and receiver device were transformed into different strains. Generator device plasmid consists of lac promoter upstream and pchBA gene downstream, so salicylate will be synthesized in the presence of IPTG.
-PchBA which locates in generator plasmid encodes an isochorismate pyruvate-lyase and an isochorismate synthase derived from Pseudomonas aeruginosa. PchA, an isochorismate synthase, catalyzes the conversion of chorismate to isochorismate. The enzyme PchB, catalyzes the conversion of isochorismate to salicylate.
-NahR is a polypeptide which plays an important role in salicylate operon. The polypeptide can exerts its salicylate-dependent activation sal operon by interacting with the promoter sequence in the region of -83 to -45 base pairs before the transcription start site. Binding of the inducer to the central domain of NahR is thought to cause additional interactions of NahR with sequences near the -35 and DNA bending, both of which promote transcriptional activation, therefore, so if there are no salicylate in the liquid nutrient medium, NahR will not binding on the promoter and lead to RNA ploymerase can not work normally, otherwise, transcription will be actived and downstream of the promoter will be transcripted, in our case, GFP will be expressed.
-<center>[[File:LM4a.png|340px]][[File:LM4b.png|340px]]</center>
-<center>(A)                                                   (B)</center>
-''Figure 4. Scheme of salicylate system plasmids and mechanism of generator and receiver system. (A) Autoinducer generator plasmid. Core elements are plac promoter, protein binding site(RBS) B0034 and autoinducer synthase gene pchA and pchB. (B) Autoinducer receiver system plasmid. This plasmid contains 3 core parts:NahR, salicylate promoter psal and sal activated gfp. (C) mechanism of experiment system. IPTG with the help of lacI could induce the expression of Protein PchA,PchB. Then they synthesize the autoinducer. In receiver system the autoinducer binds to its receiver protein CinR and the complex actived GFP expression.
-''
-== Results ==
-=== Salicylate Testing experiment ===
-In order to comfirm the system work or not. We put generator and recevier cultivate together, then centrifuged cells and resuspended them in phosphate buffer solution (PBS)(Figure.5)
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