Team:SJTU-BioX-Shanghai/Project/project1.3

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(RNA Signal)
(RNA Signal)
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===RNA Signal===
===RNA Signal===
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[[Image:12SJTU RNASIGNAL.jpg|thumb|400px|center|''Fig.10'' :
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'''A''': Sketch of signal RNA molecule (called D0) which consists of PP7 and MS2 aptamer domains.
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'''B''': Through fusing PP7 and MS2 protein to our membrane device, RNA molecule in Figure A can function as a bridge to connect different proteins, thus decreasing distance between corresponding proteins.]]
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So far, ''Membrane Accelerator'' and ''Membrane Rudder'' is generally about post-translational control over metabolic flux of the host cell. To connect this relatively isolated system to its genetic circuits, we employed RNA signal, which is present in cytoplasm. When certain RNA molecule with dimerization domain is present in cells, its cognate binding proteins can thus dimerize with each other, accelerating certain pathway.  
So far, ''Membrane Accelerator'' and ''Membrane Rudder'' is generally about post-translational control over metabolic flux of the host cell. To connect this relatively isolated system to its genetic circuits, we employed RNA signal, which is present in cytoplasm. When certain RNA molecule with dimerization domain is present in cells, its cognate binding proteins can thus dimerize with each other, accelerating certain pathway.  
More strikingly, by now, if we want to assemble certain proteins through external signal, we need to find corresponding signal-induced dimer or oligomer. But if we place RNA D0 (with  PP7 and MS2 aptamer domains) under control of various promoters regulated by different signals, approaches to induce dimerization would be expanded sharply. Thus, ''Membrane Rudder'' could sense much more signals.
More strikingly, by now, if we want to assemble certain proteins through external signal, we need to find corresponding signal-induced dimer or oligomer. But if we place RNA D0 (with  PP7 and MS2 aptamer domains) under control of various promoters regulated by different signals, approaches to induce dimerization would be expanded sharply. Thus, ''Membrane Rudder'' could sense much more signals.
We have constructed the PP7 and MS2 standardized BioBrick part, [http://partsregistry.org/Part:BBa_K771111 Part:BBa_K771111] and [http://partsregistry.org/Part:BBa_K771112 Part:BBa_K771112], respectively.
We have constructed the PP7 and MS2 standardized BioBrick part, [http://partsregistry.org/Part:BBa_K771111 Part:BBa_K771111] and [http://partsregistry.org/Part:BBa_K771112 Part:BBa_K771112], respectively.
 +
 +
[[Image:12SJTU RNASIGNAL.jpg|thumb|400px|center|''Fig.10'' :
 +
<br>
 +
'''A''': Sketch of signal RNA molecule (called D0) which consists of PP7 and MS2 aptamer domains.
 +
<br>
 +
'''B''': Through fusing PP7 and MS2 aptamer binding protein to our membrane device, RNA molecule in Figure A can function as a bridge to connect different proteins, thus decreasing distance between corresponding proteins.]]
===Chemical & Peptide Signal===
===Chemical & Peptide Signal===

Revision as of 13:43, 24 October 2012

Membrane Rudder

Dynamically and artificially regulating the direction of biochemical pathway in vivo has remained a challenge for scientists. We were trying to achieve this goal through controlling the aggregation state of different enzymes. If we replace certain constitutively dimerizing proteins in Membrane Accelerator with signal-induced dimers, then it is possible to dynamically control the direction of branched reactions.

Here is a simplified example. As is described in Figure 1, when signal that induces the aggregation of Enzyme 1 and 2 is present, the two enzymes would get close to each other, making product D more dominant. On the contrary, when signal that induces the aggregation of Enzyme 1 and 3 is present, Enzyme 1 and 3 get close to each other, so product C is more dominant.

There are indeed many signal-induced dimers that commonly exist in nature, which could sense light, chemical, peptide and even RNA signal.

Fig.1 :Demonstration of branched reactions.

Potential Signal Pool

Light Signal

Fig.2 :Dark state VVD dimers, from PDB ID 2PD7

Light is an intriguing signal to regulate E.coli activity because it is easy to obtain, highly tunable and nontoxic. A metabolite-coupled light-switchable system could be quite fascinating.

Vivid(VVD) protein, a photoreceptor of Neurospora crassa can form dimer in the presence of blue light and disassociate as light is off. Besides, VVD protein belongs to the Per-Arnt-Sim(PAS) protein superfamily.

Compared with wildtype VVD, VVD mutant (C71V and N56K), Part:BBa_K771109, is harder to dimerize in the dark and easier to dimerize under blue light . So this mutant is ideal as signal sensor in Membrane Rudder device. Membrane Anchors combined with VVD are Part:BBa_K771005 and Part:BBa_K771006, named Membrane Anchor 5 and 6 respectively.

Whether blue light is present or not could determine which enzymes should be assembled to make their corresponding product dominate.

RNA Signal

So far, Membrane Accelerator and Membrane Rudder is generally about post-translational control over metabolic flux of the host cell. To connect this relatively isolated system to its genetic circuits, we employed RNA signal, which is present in cytoplasm. When certain RNA molecule with dimerization domain is present in cells, its cognate binding proteins can thus dimerize with each other, accelerating certain pathway.

More strikingly, by now, if we want to assemble certain proteins through external signal, we need to find corresponding signal-induced dimer or oligomer. But if we place RNA D0 (with PP7 and MS2 aptamer domains) under control of various promoters regulated by different signals, approaches to induce dimerization would be expanded sharply. Thus, Membrane Rudder could sense much more signals. We have constructed the PP7 and MS2 standardized BioBrick part, Part:BBa_K771111 and Part:BBa_K771112, respectively.

Fig.10 :
A: Sketch of signal RNA molecule (called D0) which consists of PP7 and MS2 aptamer domains.
B: Through fusing PP7 and MS2 aptamer binding protein to our membrane device, RNA molecule in Figure A can function as a bridge to connect different proteins, thus decreasing distance between corresponding proteins.

Chemical & Peptide Signal

There are many ligand-induced dimers in nature. ErbB family proteins are typically ligand-induced dimers. Estrogen receptor only forms dimer when estrogen is present. If incorporated into our system, it might have significant application prospect. BMP receptors could form heterodimer in the presence of their cognate ligand.

Many other receptors or transport proteins could also form chemical-signal induced dimers.

Recruiting those chemical signal induced dimers could easily broaden the application field of our system.

Application

What we want to offer is a universal tool which can be connected to different downstream enzymes and upstream signal sensors. We chose blue light sensor protein -- VVD to test the feasibility of Membrane Rudder device because of the uniqueness of light signal. Light signal is easily obtained,

Violacein and deoxyviolacein synthetic pathway, a branched enzymatic reaction is recruited to prove Membrane Rudder sensitive to blue light could work as expected.

Reference

1.Delebecque, C. J., A. B. Lindner, et al. (2011). "Organization of intracellular reactions with rationally designed RNA assemblies." Science 333(6041): 470.

2.Kumar, V. and P. Chambon (1988). "The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer." Cell 55(1): 145.

3.Lemmon, M. A. (2009). "Ligand-induced ErbB receptor dimerization." Experimental cell research 315(4): 638-648.

4.Terrillon, S. and M. Bouvier (2004). "Roles of G-protein-coupled receptor dimerization." EMBO reports 5(1): 30-34.

5.Wang, X., X. Chen, et al. (2012). "Spatiotemporal control of gene expression by a light-switchable transgene system." Nature Methods.