IGEM:Harvard/2006/Brainstorming Papers - Perry Tsai

Quorum sensing

 * 1) qs1 pmid=16330045

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.

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.
 * 1) qs2 pmid=16467145

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.

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.
 * 1) qs3 pmid=15256602


 * 1) qs4 pmid=15159530
 * 2) qs5 pmid=15130116
 * 3) qs6 pmid=15064770
 * 4) qs7 pmid=11929534

DNA aptamers
Functional nucleic acids, or FNAs.
 * 1) dnaA1 pmid=16678470
 * 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.

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.
 * 1) dnaA2 pmid=16283295
 * 2) dnaA3 pmid=16199173
 * 3) dnaA4 pmid=16146351
 * 4) dnaA5 pmid=16117506

Ion channels/transporters
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.
 * 1) ion1 pmid=16574149
 * 2) ion2 pmid=16525509
 * 3) ion3 pmid=16484207
 * 4) ion4 pmid=16413498

Proton gradient can be coupled to drive EmrE, a proton-coupled exporter of lipophilic toxic cations, like ethidium. Measured by fluorescence of ethidium.

ChaA mediates K+ efflux against K+ concentration gradient, discards excessive K+ which would be toxic. 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.
 * 1) ion5 pmid=16390457
 * 2) ion6 pmid=16316975
 * 3) ion7 pmid=16687400
 * 1) ion8 pmid=12384697