一种可实现合成生物传感器现场部署的增材制造方法
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摘要
合成生物学工具可用于设计活体生物传感器,报告目标分析物的存在。虽然这些工程细胞生物传感器在实验室外具有许多潜在应用,但由于它们属于转基因生物(genetically modified organism,GMO),通常被认为具有危险性。因此,如何在实验室外使用转基因生物的同时,降低将其释放到环境中的风险就至关重要。本文描述了一种包含合成生物电路的生物传感系统。含有该系统的大肠杆菌(Escherichia coli)被置于一个特制的3D打印的试管盖内。这些转基因生物能够检测到一种条件致病菌铜绿假单胞菌(Pseudomonas aeruginosa)的化学群体信号。在该设备中,活体生物传感器可以在不接触环境的情况下,接触感兴趣的样本。细胞可以在培养管内进行现场可视化分析,也可以送回实验室进行进一步分析。许多生物传感器缺乏现场部署所需的多功能性,由于缺乏资源和装置,许多疾病可能无法诊断。我们的生物检测设备利用3D打印技术,为现场部署活体生物传感器制造了一种便携式、模块化和廉价的设备。
The tools of synthetic biology can be used to engineer living biosensors that report the presence of analytes. Although these engineered cellular biosensors have many potential applications for deployment outside of the lab, they are genetically modified organisms(GMOs) and are often considered dangerous.Mitigating the risk of releasing GMOs into the environment while enabling their use outside a laboratory is critical. Here, we describe the development of a biosensing system consisting of a synthetic biological circuit, which is engineered in Escherichia coli that are contained within a unique 3 D-printed device housing. These GMOs detect the chemical quorum signal of Pseudomonas aeruginosa, an opportunistic pathogen. Using this device, the living biosensor makes contact with a specimen of interest without ever being exposed to the environment. Cells can be visually analyzed in the field within culture tubes, or returned to the lab for further analysis. Many biosensors lack the versatility required for deployment in the field, where many diseases can go undiagnosed due to a lack of resources and equipment. Our bioassay device utilizes 3 D printing to create a portable, modular, and inexpensive device for the field deployment of living biosensors.
The tools of synthetic biology can be used to engineer living biosensors that report the presence of analytes. Although these engineered cellular biosensors have many potential applications for deployment outside of the lab, they are genetically modified organisms(GMOs) and are often considered dangerous.Mitigating the risk of releasing GMOs into the environment while enabling their use outside a laboratory is critical. Here, we describe the development of a biosensing system consisting of a synthetic biological circuit, which is engineered in Escherichia coli that are contained within a unique 3 D-printed device housing. These GMOs detect the chemical quorum signal of Pseudomonas aeruginosa, an opportunistic pathogen. Using this device, the living biosensor makes contact with a specimen of interest without ever being exposed to the environment. Cells can be visually analyzed in the field within culture tubes, or returned to the lab for further analysis. Many biosensors lack the versatility required for deployment in the field, where many diseases can go undiagnosed due to a lack of resources and equipment. Our bioassay device utilizes 3 D printing to create a portable, modular, and inexpensive device for the field deployment of living biosensors.
引文
[1]Gardner TS,Cantor CR,Collins JJ.Construction of a genetic toggle switch in Escherichia coli.Nature 2000;403(6767):339-42.
[2]Elowitz MB,Leibler S.A synthetic oscillatory network of transcriptional regulators.Nature 2000;403(6767):335-8.
[3]BalagaddéFK,Song H,Ozaki J,Collins CH,Barnet M,Arnold FH,et al.Asynthetic Escherichia coli predator-prey ecosystem.Mol Syst Biol 2008;4(1):187.
[4]Litcofsky KD,Afeyan RB,Krom RJ,Khalil AS,Collins JJ.Iterative plug-and-play methodology for constructing and modifying synthetic gene networks.Nat Methods 2012;9(11):1077-80.
[5]Brenner K,Karig DK,Weiss R,Arnold FH.Engineered bidirectional communication mediates a consensus in a microbial biofilm consortium.Proc Natl Acad Sci USA 2007;104(44):17300-4.
[6]Tamsir A,Tabor JJ,Voigt CA.Robust multicellular computing using genetically encoded NOR gates and chemical‘wires’.Nature 2011;469(7329):212-5.
[7]McDaniel LE,Bailey EG,Zimmerli A.Effect of oxygen-supply rates on growth of Escherichia coli.I.Studies in unbaffled and baffled shake flasks.Appl Microbiol1965;13:109-14.
[8]Ratkowsky DA,Olley J,McMeekin TA,Ball A.Relationship between temperature and growth rate of bacterial cultures.J Bacteriol 1982;149(1):1-5.
[9]Kuzma J,Besley JC.Ethics of risk analysis and regulatory review:from bio-to nanotechnology.NanoEthics 2008;2(2):149-62.
[10]Gregorowius D,Lindemann-Matthies P,Huppenbauer M.Ethical discourse on the use of genetically modified crops:a review of academic publications in the fields of ecology and environmental ethics.J Agric Environ Ethics 2012;25(3):265-93.
[11]Kilama WL.Health research ethics in public health:trials and implementation of malaria mosquito control strategies.Acta Trop 2009;112(Suppl 1):S37-47.
[12]Schmidt M.Special issue:societal aspects of synthetic biology.Syst Synth Biol2009;3(1-4):1-2.
[13]Kuzma J,Tanji T.Unpackaging synthetic biology:identification of oversight policy problems and options.Regul Governance 2010;4(1):92-112.
[14]Melchels FPW,Feijen J,Grijpma DW.A review on stereolithography and its applications in biomedical engineering.Biomaterials 2010;31(24):6121-30.
[15]Dimitrov D,Schreve K,De Beer N.Advances in three dimensional printingstate of the art and future perspectives.Rapid Prototyping J 2006;12(3):136-47.
[16]Schubert C,Van Langeveld MC,Donoso LA.Innovations in 3D printing:a 3Doverview from optics to organs.Br J Ophthalmol 2014;98(2):159-61.
[17]Ventola CL.Medical applications for 3D printing:current and projected uses.PT 2014;39(10):704-11.
[18]Gambello MJ,Iglewski BH.Cloning and characterization of the Pseudomonas aeruginosa lasR gene,a transcriptional activator of elastase expression.JBacteriol 1991;173(9):3000-9.
[19]Kiratisin P,Tucker KD,Passador L.LasR,a transcriptional activator of Pseudomonas aeruginosa virulence genes,functions as a multimer.J Bacteriol2002;184(17):4912-9.
[20]Schwarzer C,Fu Z,Fischer H,Machen TE.Redox-independent activation of NF-j B by Pseudomonas aeruginosa pyocyanin in a cystic fibrosis airway epithelial cell line.J Biol Chem 2008;283(40):27144-53.
[21]Pearson JP,Pesci EC,Iglewski BH.Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes.J Bacteriol 1997;179(18):5756-67.
[22]Seed PC,Passador L,Iglewski BH.Activation of the Pseudomonas aeruginosa lasIgene by LasR and the Pseudomonas autoinducer PAI:an autoinduction regulatory hierarchy.J Bacteriol 1995;177(3):654-9.
[23]Lyczak JB,Cannon CL,Pier GB.Lung infections associated with cystic fibrosis.Clin Microbiol Rev 2002;15(2):194-222.
[24]MaciáMD,Blanquer D,Togores B,Sauleda J,Pérez JL,Oliver A.Hypermutation is a key factor in development of multiple-antimicrobial resistance in Pseudomonas aeruginosa strains causing chronic lung infections.Antimicrob Agents Chemother 2005;49(8):3382-6.
[25]Pier GB,Grout M,Zaidi TS,Olsen JC,Johnson LG,Yankaskas JR,et al.Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections.Science 1996;271(5245):64-7.
[26]Pedersen SS,Espersen F,H?iby N.Diagnosis of chronic Pseudomonas aeruginosa infection in cystic fibrosis by enzyme-linked immunosorbent assay.J Clin Microbiol 1987;25(10):1830-6.
[27]McCulloch E,Lucas C,Ramage G,Williams C.Improved early diagnosis of Pseudomonas aeruginosa by real-time PCR to prevent chronic colonisation in a paediatric cystic fibrosis population.J Cyst Fibros 2011;10(1):21-4.
[28]Pearson JP,Gray KM,Passador L,Tucker KD,Eberhard A,Iglewski BH,et al.Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes.Proc Natl Acad Sci USA 1994;91(1):197-201.
[29]Maniatis T,Fritsch EF,Sambrook J.Molecular cloning:a laboratory manual.2nd ed.New York:Cold Spring Harbor Laboratory Press;1989.
[30]Ausubel FM,Brent R,Kingston R,Moore DD,Seidman J,Struhl K,et al.Current protocols in molecular biology.New York:John Wiley&Sons;1987.
[31]Brophy JA,Voigt CA.Principles of genetic circuit design.Nat Methods 2014;11(5):508-20.
[32]Hindmarsh AC.LSODE and LSODI,two new initial value ordinary differential equation solvers.ACM-SIGNUM Newslett 1980;15(4):10-1.
[33]COMSOL.COMSOL multiphysics:user’s guide(version 4.3 a).Stockholm:COMSOL;2012.
[34]Szykiedans K,Credo W.Mechanical properties of FDM and SLA low-cost 3-Dprints.Procedia Eng 2016;136:257-62.
[35]Pardee K,Green AA,Ferrante T,Cameron DE,DaleyKeyser A,Yin P,et al.Paperbased synthetic gene networks.Cell 2014;159(4):940-54.
[36]Dubin PJ,Kolls JK.IL-23 mediates inflammatory responses to mucoid Pseudomonas aeruginosa lung infection in mice.Am J Physiol Lung Cell Mol Physiol 2007;292(2):L519-28.
[37]Jensen T,Pedersen SS,Garne S,Heilmann C,H?iby N,Koch C.Colistin inhalation therapy in cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection.J Antimicrob Chemother 1987;19(6):831-8.
[38]Ciofu O,Riis B,Pressler T,Poulsen HE,H?iby N.Occurrence of hypermutable Pseudomonas aeruginosa in cystic fibrosis patients is associated with the oxidative stress caused by chronic lung inflammation.Antimicrob Agents Chemother 2005;49(6):2276-82.
[39]Heeckeren A,Walenga R,Konstan MW,Bonfield T,Davis PB,Ferkol T.Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa.J Clin Invest 1997;100(11):2810-5.
[40]Jahoor A,Patel R,Bryan A,Do C,Krier J,Watters C,et al.Peroxisome proliferator-activated receptors mediate host cell proinflammatory responses to Pseudomonas aeruginosa autoinducer.J Bacteriol 2008;190(13):4408-15.
[41]Kruger NJ.The Bradford method for protein quantitation.In:Walker JM,editor.Basic protein and peptide protocols.New York:Humana Press;1994.p.9-15.
[42]Swanson AB,Matev IB,de Groot G.The strength of the hand.Bull Prosthet Res1970;10(14):145-53.
[2]Elowitz MB,Leibler S.A synthetic oscillatory network of transcriptional regulators.Nature 2000;403(6767):335-8.
[3]BalagaddéFK,Song H,Ozaki J,Collins CH,Barnet M,Arnold FH,et al.Asynthetic Escherichia coli predator-prey ecosystem.Mol Syst Biol 2008;4(1):187.
[4]Litcofsky KD,Afeyan RB,Krom RJ,Khalil AS,Collins JJ.Iterative plug-and-play methodology for constructing and modifying synthetic gene networks.Nat Methods 2012;9(11):1077-80.
[5]Brenner K,Karig DK,Weiss R,Arnold FH.Engineered bidirectional communication mediates a consensus in a microbial biofilm consortium.Proc Natl Acad Sci USA 2007;104(44):17300-4.
[6]Tamsir A,Tabor JJ,Voigt CA.Robust multicellular computing using genetically encoded NOR gates and chemical‘wires’.Nature 2011;469(7329):212-5.
[7]McDaniel LE,Bailey EG,Zimmerli A.Effect of oxygen-supply rates on growth of Escherichia coli.I.Studies in unbaffled and baffled shake flasks.Appl Microbiol1965;13:109-14.
[8]Ratkowsky DA,Olley J,McMeekin TA,Ball A.Relationship between temperature and growth rate of bacterial cultures.J Bacteriol 1982;149(1):1-5.
[9]Kuzma J,Besley JC.Ethics of risk analysis and regulatory review:from bio-to nanotechnology.NanoEthics 2008;2(2):149-62.
[10]Gregorowius D,Lindemann-Matthies P,Huppenbauer M.Ethical discourse on the use of genetically modified crops:a review of academic publications in the fields of ecology and environmental ethics.J Agric Environ Ethics 2012;25(3):265-93.
[11]Kilama WL.Health research ethics in public health:trials and implementation of malaria mosquito control strategies.Acta Trop 2009;112(Suppl 1):S37-47.
[12]Schmidt M.Special issue:societal aspects of synthetic biology.Syst Synth Biol2009;3(1-4):1-2.
[13]Kuzma J,Tanji T.Unpackaging synthetic biology:identification of oversight policy problems and options.Regul Governance 2010;4(1):92-112.
[14]Melchels FPW,Feijen J,Grijpma DW.A review on stereolithography and its applications in biomedical engineering.Biomaterials 2010;31(24):6121-30.
[15]Dimitrov D,Schreve K,De Beer N.Advances in three dimensional printingstate of the art and future perspectives.Rapid Prototyping J 2006;12(3):136-47.
[16]Schubert C,Van Langeveld MC,Donoso LA.Innovations in 3D printing:a 3Doverview from optics to organs.Br J Ophthalmol 2014;98(2):159-61.
[17]Ventola CL.Medical applications for 3D printing:current and projected uses.PT 2014;39(10):704-11.
[18]Gambello MJ,Iglewski BH.Cloning and characterization of the Pseudomonas aeruginosa lasR gene,a transcriptional activator of elastase expression.JBacteriol 1991;173(9):3000-9.
[19]Kiratisin P,Tucker KD,Passador L.LasR,a transcriptional activator of Pseudomonas aeruginosa virulence genes,functions as a multimer.J Bacteriol2002;184(17):4912-9.
[20]Schwarzer C,Fu Z,Fischer H,Machen TE.Redox-independent activation of NF-j B by Pseudomonas aeruginosa pyocyanin in a cystic fibrosis airway epithelial cell line.J Biol Chem 2008;283(40):27144-53.
[21]Pearson JP,Pesci EC,Iglewski BH.Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes.J Bacteriol 1997;179(18):5756-67.
[22]Seed PC,Passador L,Iglewski BH.Activation of the Pseudomonas aeruginosa lasIgene by LasR and the Pseudomonas autoinducer PAI:an autoinduction regulatory hierarchy.J Bacteriol 1995;177(3):654-9.
[23]Lyczak JB,Cannon CL,Pier GB.Lung infections associated with cystic fibrosis.Clin Microbiol Rev 2002;15(2):194-222.
[24]MaciáMD,Blanquer D,Togores B,Sauleda J,Pérez JL,Oliver A.Hypermutation is a key factor in development of multiple-antimicrobial resistance in Pseudomonas aeruginosa strains causing chronic lung infections.Antimicrob Agents Chemother 2005;49(8):3382-6.
[25]Pier GB,Grout M,Zaidi TS,Olsen JC,Johnson LG,Yankaskas JR,et al.Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections.Science 1996;271(5245):64-7.
[26]Pedersen SS,Espersen F,H?iby N.Diagnosis of chronic Pseudomonas aeruginosa infection in cystic fibrosis by enzyme-linked immunosorbent assay.J Clin Microbiol 1987;25(10):1830-6.
[27]McCulloch E,Lucas C,Ramage G,Williams C.Improved early diagnosis of Pseudomonas aeruginosa by real-time PCR to prevent chronic colonisation in a paediatric cystic fibrosis population.J Cyst Fibros 2011;10(1):21-4.
[28]Pearson JP,Gray KM,Passador L,Tucker KD,Eberhard A,Iglewski BH,et al.Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes.Proc Natl Acad Sci USA 1994;91(1):197-201.
[29]Maniatis T,Fritsch EF,Sambrook J.Molecular cloning:a laboratory manual.2nd ed.New York:Cold Spring Harbor Laboratory Press;1989.
[30]Ausubel FM,Brent R,Kingston R,Moore DD,Seidman J,Struhl K,et al.Current protocols in molecular biology.New York:John Wiley&Sons;1987.
[31]Brophy JA,Voigt CA.Principles of genetic circuit design.Nat Methods 2014;11(5):508-20.
[32]Hindmarsh AC.LSODE and LSODI,two new initial value ordinary differential equation solvers.ACM-SIGNUM Newslett 1980;15(4):10-1.
[33]COMSOL.COMSOL multiphysics:user’s guide(version 4.3 a).Stockholm:COMSOL;2012.
[34]Szykiedans K,Credo W.Mechanical properties of FDM and SLA low-cost 3-Dprints.Procedia Eng 2016;136:257-62.
[35]Pardee K,Green AA,Ferrante T,Cameron DE,DaleyKeyser A,Yin P,et al.Paperbased synthetic gene networks.Cell 2014;159(4):940-54.
[36]Dubin PJ,Kolls JK.IL-23 mediates inflammatory responses to mucoid Pseudomonas aeruginosa lung infection in mice.Am J Physiol Lung Cell Mol Physiol 2007;292(2):L519-28.
[37]Jensen T,Pedersen SS,Garne S,Heilmann C,H?iby N,Koch C.Colistin inhalation therapy in cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection.J Antimicrob Chemother 1987;19(6):831-8.
[38]Ciofu O,Riis B,Pressler T,Poulsen HE,H?iby N.Occurrence of hypermutable Pseudomonas aeruginosa in cystic fibrosis patients is associated with the oxidative stress caused by chronic lung inflammation.Antimicrob Agents Chemother 2005;49(6):2276-82.
[39]Heeckeren A,Walenga R,Konstan MW,Bonfield T,Davis PB,Ferkol T.Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa.J Clin Invest 1997;100(11):2810-5.
[40]Jahoor A,Patel R,Bryan A,Do C,Krier J,Watters C,et al.Peroxisome proliferator-activated receptors mediate host cell proinflammatory responses to Pseudomonas aeruginosa autoinducer.J Bacteriol 2008;190(13):4408-15.
[41]Kruger NJ.The Bradford method for protein quantitation.In:Walker JM,editor.Basic protein and peptide protocols.New York:Humana Press;1994.p.9-15.
[42]Swanson AB,Matev IB,de Groot G.The strength of the hand.Bull Prosthet Res1970;10(14):145-53.
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