IGEM:Harvard/2006/Brainstorming Papers - Perry Tsai
- Anderson JC, Clarke EJ, Arkin AP, and Voigt CA. Environmentally controlled invasion of cancer cells by engineered bacteria. J Mol Biol. 2006 Jan 27;355(4):619-27. DOI:10.1016/j.jmb.2005.10.076 |
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.
- Yang S, Lopez CR, and Zechiedrich EL. Quorum sensing and multidrug transporters in Escherichia coli. Proc Natl Acad Sci U S A. 2006 Feb 14;103(7):2386-91. DOI:10.1073/pnas.0502890102 |
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.
- Garcia-Ojalvo J, Elowitz MB, and Strogatz SH. Modeling a synthetic multicellular clock: repressilators coupled by quorum sensing. Proc Natl Acad Sci U S A. 2004 Jul 27;101(30):10955-60. DOI:10.1073/pnas.0307095101 |
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.
- Kobayashi H, Kaern M, Araki M, Chung K, Gardner TS, Cantor CR, and Collins JJ. Programmable cells: interfacing natural and engineered gene networks. Proc Natl Acad Sci U S A. 2004 Jun 1;101(22):8414-9. DOI:10.1073/pnas.0402940101 |
- Ahmer BM. Cell-to-cell signalling in Escherichia coli and Salmonella enterica. Mol Microbiol. 2004 May;52(4):933-45. DOI:10.1111/j.1365-2958.2004.04054.x |
- You L, Cox RS 3rd, Weiss R, and Arnold FH. Programmed population control by cell-cell communication and regulated killing. Nature. 2004 Apr 22;428(6985):868-71. DOI:10.1038/nature02491 |
- Sperandio V, Torres AG, and Kaper JB. Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli. Mol Microbiol. 2002 Feb;43(3):809-21.
- Navani NK and Li Y. Nucleic acid aptamers and enzymes as sensors. Curr Opin Chem Biol. 2006 Jun;10(3):272-81. DOI:10.1016/j.cbpa.2006.04.003 |
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.
- Proske D, Blank M, Buhmann R, and Resch A. Aptamers--basic research, drug development, and clinical applications. Appl Microbiol Biotechnol. 2005 Dec;69(4):367-74. DOI:10.1007/s00253-005-0193-5 |
- Nutiu R and Li Y. Aptamers with fluorescence-signaling properties. Methods. 2005 Sep;37(1):16-25. DOI:10.1016/j.ymeth.2005.07.001 |
- Patil SD, Rhodes DG, and Burgess DJ. DNA-based therapeutics and DNA delivery systems: a comprehensive review. AAPS J. 2005 Apr 8;7(1):E61-77. DOI:10.1208/aapsj070109 |
- So HM, Won K, Kim YH, Kim BK, Ryu BH, Na PS, Kim H, and Lee JO. Single-walled carbon nanotube biosensors using aptamers as molecular recognition elements. J Am Chem Soc. 2005 Aug 31;127(34):11906-7. DOI:10.1021/ja053094r |
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.
- Vadyvaloo V, Smirnova IN, Kasho VN, and Kaback HR. Conservation of residues involved in sugar/H(+) symport by the sucrose permease of Escherichia coli relative to lactose permease. J Mol Biol. 2006 May 12;358(4):1051-9. DOI:10.1016/j.jmb.2006.02.050 |
- Mirza O, Guan L, Verner G, Iwata S, and Kaback HR. Structural evidence for induced fit and a mechanism for sugar/H+ symport in LacY. EMBO J. 2006 Mar 22;25(6):1177-83. DOI:10.1038/sj.emboj.7601028 |
- Rothenbücher MC, Facey SJ, Kiefer D, Kossmann M, and Kuhn A. The cytoplasmic C-terminal domain of the Escherichia coli KdpD protein functions as a K+ sensor. J Bacteriol. 2006 Mar;188(5):1950-8. DOI:10.1128/JB.188.5.1950-1958.2006 |
- Kamo N, Hashiba T, Kikukawa T, Araiso T, Ihara K, and Nara T. A light-driven proton pump from Haloterrigena turkmenica: functional expression in Escherichia coli membrane and coupling with a H+ co-transporter. Biochem Biophys Res Commun. 2006 Mar 10;341(2):285-90. DOI:10.1016/j.bbrc.2005.12.181 |
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.
- Radchenko MV, Waditee R, Oshimi S, Fukuhara M, Takabe T, and Nakamura T. Cloning, functional expression and primary characterization of Vibrio parahaemolyticus K+/H+ antiporter genes in Escherichia coli. Mol Microbiol. 2006 Jan;59(2):651-63. DOI:10.1111/j.1365-2958.2005.04966.x |
- Accardi A, Walden M, Nguitragool W, Jayaram H, Williams C, and Miller C. Separate ion pathways in a Cl-/H+ exchanger. J Gen Physiol. 2005 Dec;126(6):563-70. DOI:10.1085/jgp.200509417 |
- Radchenko MV, Tanaka K, Waditee R, Oshimi S, Matsuzaki Y, Fukuhara M, Kobayashi H, Takabe T, and Nakamura T. Potassium/proton antiport system of Escherichia coli. J Biol Chem. 2006 Jul 21;281(29):19822-9. DOI:10.1074/jbc.M600333200 |
ChaA mediates K+ efflux against K+ concentration gradient, discards excessive K+ which would be toxic.
- Iyer R, Iverson TM, Accardi A, and Miller C. A biological role for prokaryotic ClC chloride channels. Nature. 2002 Oct 17;419(6908):715-8. DOI:10.1038/nature01000 |
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.