IGEM:Harvard/2006/Brainstorming Papers - Perry Tsai

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Quorum sensing

  1. 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 | PubMed ID:16330045 | HubMed [qs1]

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.

  1. 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 | PubMed ID:16467145 | HubMed [qs2]

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.

  1. 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 | PubMed ID:15256602 | HubMed [qs3]

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. 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 | PubMed ID:15159530 | HubMed [qs4]
  2. 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 | PubMed ID:15130116 | HubMed [qs5]
  3. 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 | PubMed ID:15064770 | HubMed [qs6]
  4. 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. PubMed ID:11929534 | HubMed [qs7]

All Medline abstracts: PubMed | HubMed

DNA aptamers

  1. 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 | PubMed ID:16678470 | HubMed [dnaA1]

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.
  1. 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 | PubMed ID:16283295 | HubMed [dnaA2]
  2. 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 | PubMed ID:16199173 | HubMed [dnaA3]
  3. 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 | PubMed ID:16146351 | HubMed [dnaA4]
  4. 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 | PubMed ID:16117506 | HubMed [dnaA5]

All Medline abstracts: PubMed | HubMed

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.

Ion channels/transporters

  1. 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 | PubMed ID:16574149 | HubMed [ion1]
  2. 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 | PubMed ID:16525509 | HubMed [ion2]
  3. Rothenb├╝cher 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 | PubMed ID:16484207 | HubMed [ion3]
  4. 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 | PubMed ID:16413498 | HubMed [ion4]

All Medline abstracts: PubMed | HubMed

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.

  1. 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 | PubMed ID:16390457 | HubMed [ion5]
  2. 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 | PubMed ID:16316975 | HubMed [ion6]
  3. 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 | PubMed ID:16687400 | HubMed [ion7]

All Medline abstracts: PubMed | HubMed

ChaA mediates K+ efflux against K+ concentration gradient, discards excessive K+ which would be toxic.

  1. 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 | PubMed ID:12384697 | HubMed [ion8]

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.