IGEM:Harvard/2006/Brainstorming Papers - Perry Tsai
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inv gene encoding invasin from Yersinia pseudotuberculosis initiates adhesion and invasion of E.coli into beta1-integrin-expressing mammalian cells, without the need for other adhesion or invasion machinery. This is linked to cell density by linking inv to quorum-sensing lux operon. They also created arabinose and hypoxia inducible versions through genetic selection. Certain bacterial speicies localize to tumors.
Quorum-sensing. The circuit encodes transcriptional activator LuxR and enzyme LuxI. LuxI catalyses synthesis of AI-1 which diffuses into media. At high density, AI-1 activates luxR which in turn upregulates luxI and luxR. This causes rapid state change. inv was fused with luxPr promoter downstream of luxI.
Application? Switch to turn on synthesis of a chemotherapeutic prodrug at tumor sites. Synthesis of antigens to act as vaccines. Gene delivery vector.
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acrAB-, norE-, acrAB-/norE-, but not mdfA- allows E. coli cultures to grow to a greater density in stationary phase. Conditioned medium from acrAB-/norE- allows more growth in stationary phase. CM from cells overexpressing acrAB or norE repress growth in stationary phase.
Proposal: AcrAB, NorE, and other MDR pumps promote cell-cell communication by extruding quorum-sensing signals more efficiently than the signals can diffuse on their own. It's unknown what the QSS is; could resemble fluoroquinolone class of antibiotics.
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They coupled Elowitz and Leibler's cI-|lacI-|tetR-|cI repressilator and linked it to quorum-sensing molecules. The genetic circuit concept was that, in addition to repressing tetR, lacI would also repress the expression of luxI, which codes from protein AI, autoinducer, a quorum-sensing molecule that is diffusible across the membrane. The AI-LuxR complex would be engineered to activate expression of a second copy of another repressilator gene, like LacI. The result was synchronized oscillators.
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Functional nucleic acids, or FNAs.
- Optical sensing. Using fluorescently labelled FNAs. Molecular beacon: hairpin which joins a fluorophore and a quencher, binding causes opening of hairpin and separation of F and Q. Duplex-to-complex approach is same but an F-labelled aptamer with complementary Q-labelled strand.
- Acoustic sensing. Mass changes measured on quartz vua surface-acoustic wave. Has been shown to detect human thrombin and HIV-1 Tat.
- Cantilever-based sensing. Cantilever bound to aptamer; binding of target causes mechanial signal.
- Electrochemical signalling. Negatively charged aptamer prevents redox at an electrode. Binding of a positively charged protein reduces negative charge, this lowering electron transfer resistance. Or a hairpin that binds methylene blue, which is an electrochemical signal; binding opens hairpin, releases methylene blue.
- Many studies weaken an existing nucleic acid enzyme; target binding restores full activity. For example, a hairpin blocking the catalytic site of a deoxyribozyme. Or two parts of a catalysis are brought togehter by binding to the same nucleir acid target. Or an thrombin-binding DNA aptamer inhibits thrombin activity; binding of nucleic acid target causes opening of thrombin to cleave a fluorogenic peptide substrate.
- Aptamers can also bind metabolites (ATP, cAMP) and metal ions (lead, mercury).
- Gold nanoparticles are red when isolated, blue when grouped. Gold can bind through DNA oligos to aptamer (aggregate, blue); addition of target turns on deoxyribozyme, disassembles, gold shows red.
- Carbon nanotubes have high mechanical strength and can be insulating, semiconducting, or conducting. Binding to aptamer changes conductance.
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They created a biosensor of thrombin by attaching a thrombin DNA aptamer to a carbon nanotube via CDI-Tween. The successful binding of thrombin was indicated by a drop in conductance.
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Expressed functional HtdR (H. turkmenica deltarhodopsin) in E. coli. Bacteriorhodopsin in H. salinarum is light-driven and transfers one proton from cytoplasm to medium. Same photocycle as deltarhodopsin.
Proton gradient can be coupled to drive EmrE, a proton-coupled exporter of lipophilic toxic cations, like ethidium. Measured by fluorescence of ethidium.
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ChaA mediates K+ efflux against K+ concentration gradient, discards excessive K+ which would be toxic.
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E. coli uses chloride channels for as extreme acid resistance response. The channels function as an electrical shunt for an outwardly directed virtual proton pump linked to aminoi acid decarboxylation.