On a Robust, Sensitive Cell-Free Method for Pseudomonas Sensing and Quantification in Microfluidic Templated Hydrogels

Seto, Jong

doi:10.3390/mi10080506

Title: On a Robust, Sensitive Cell-Free Method for Pseudomonas Sensing and Quantification in Microfluidic Templated Hydrogels

Journal Article · Wed Jul 31 00:00:00 EDT 2019 · Micromachines

DOI:https://doi.org/10.3390/mi10080506· OSTI ID:1628462

^[1]

University of California at San Francisco and California, San Francisco, CA (United States). Institute for Quantitative Biosciences (QB3), Department of Bioengineering and Therapeutic Sciences; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Foundry

Through the use of droplet microfluidics to integrate cell-free activity into inert hydrogel beads, we have developed a platform that can perform biologically relevant functions without the need for cells. Specifically, cell-free lysates serve a utility in performing cellular functions and providing biologically relevant metabolic products without requiring the optimal biological conditions for cell growth and proliferation. By teasing out specific biological components that enable transcription and translation to occur, these cell-like functions can be reconstituted in vitro without requiring the entire cell and milieu of cellular organelles. This enables the optimization of synthetic biological circuits, either by concentration or logic switches, simply through the addition or removal of genetic components (plasmids, inducers, or repressors) of regulatory elements. Here, we demonstrate an application of cell-free processes that is robust and portable, independent of a substrate, to apply for sensing and reporting functions of a quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12HSL) found crucial for pathological Pseudomonas aeruginosa infection. We develop an agarose bead platform that is easily adaptable and simply programmable to fit a variety of biological and chemical sensing applications for the utility of ease of delivery and activation in remote environments—even in conditions with very little hydration.

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Research Organization:: Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1628462

Journal Information:: Micromachines, Vol. 10, Issue 8; ISSN 2072-666X

Publisher:: MDPICopyright Statement

Country of Publication:: United States

Language:: English

References (56)

Transcriptome analysis of quorum-sensing regulation and virulence factor expression in Pseudomonas aeruginosa Wagner, Victoria E.; Gillis, Richard J.; Iglewski, Barbara H. Vaccine, Vol. 22 https://doi.org/10.1016/j.vaccine.2004.08.011	journal	December 2004
Rapid cell-free forward engineering of novel genetic ring oscillators Niederholtmeyer, Henrike; Sun, Zachary Z.; Hori, Yutaka eLife, Vol. 4 https://doi.org/10.7554/eLife.09771	journal	October 2015
Cell-free expression systems for eukaryotic protein production Endo, Yaeta; Sawasaki, Tatsuya Current Opinion in Biotechnology, Vol. 17, Issue 4 https://doi.org/10.1016/j.copbio.2006.06.009	journal	August 2006
Fluorescent Proteins and in Vitro Genetic Organization for Cell-Free Synthetic Biology Lentini, Roberta; Forlin, Michele; Martini, Laura ACS Synthetic Biology, Vol. 2, Issue 9 https://doi.org/10.1021/sb400003y	journal	March 2013
QUORUM SENSING: Cell-to-Cell Communication in Bacteria Waters, Christopher M.; Bassler, Bonnie L. Annual Review of Cell and Developmental Biology, Vol. 21, Issue 1 https://doi.org/10.1146/annurev.cellbio.21.012704.131001	journal	November 2005
Genomic mining of prokaryotic repressors for orthogonal logic gates Stanton, Brynne C.; Nielsen, Alec A. K.; Tamsir, Alvin Nature Chemical Biology, Vol. 10, Issue 2 https://doi.org/10.1038/nchembio.1411	journal	December 2013
Safeguarding CRISPR-Cas9 gene drives in yeast DiCarlo, James E.; Chavez, Alejandro; Dietz, Sven L. Nature Biotechnology, Vol. 33, Issue 12 https://doi.org/10.1038/nbt.3412	journal	November 2015
High-throughput preparation methods of crude extract for robust cell-free protein synthesis Kwon, Yong-Chan; Jewett, Michael C. Scientific Reports, Vol. 5, Issue 1 https://doi.org/10.1038/srep08663	journal	March 2015
Programmable bacteria detect and record an environmental signal in the mammalian gut Kotula, Jonathan W.; Kerns, S. Jordan; Shaket, Lev A. Proceedings of the National Academy of Sciences, Vol. 111, Issue 13 https://doi.org/10.1073/pnas.1321321111	journal	March 2014
Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression Didovyk, Andriy; Tonooka, Taishi; Tsimring, Lev ACS Synthetic Biology, Vol. 6, Issue 12 https://doi.org/10.1021/acssynbio.7b00253	journal	August 2017
Mechanically Tailored Agarose Hydrogels through Molecular Alloying with β -Sheet Polysaccharides Forget, Aurelien; Pique, Raphaelle-Anne; Ahmadi, Vincent Macromolecular Rapid Communications, Vol. 36, Issue 2 https://doi.org/10.1002/marc.201400353	journal	September 2014
Principles of cell-free genetic circuit assembly Noireaux, V.; Bar-Ziv, R.; Libchaber, A. Proceedings of the National Academy of Sciences, Vol. 100, Issue 22 https://doi.org/10.1073/pnas.2135496100	journal	October 2003
Central Dogma of Molecular Biology Crick, Francis Nature, Vol. 227, Issue 5258 https://doi.org/10.1038/227561a0	journal	August 1970
Communication between hydrogel beads via chemical signalling Jaggers, Ross W.; Bon, Stefan A. F. Journal of Materials Chemistry B, Vol. 5, Issue 44 https://doi.org/10.1039/C7TB02278F	journal	January 2017
Dynamics of CRISPR-Cas9 genome interrogation in living cells Knight, Spencer C.; Xie, Liangqi; Deng, Wulan Science, Vol. 350, Issue 6262 https://doi.org/10.1126/science.aac6572	journal	November 2015
Genetic programs constructed from layered logic gates in single cells Moon, Tae Seok; Lou, Chunbo; Tamsir, Alvin Nature, Vol. 491, Issue 7423 https://doi.org/10.1038/nature11516	journal	October 2012
A vesicle bioreactor as a step toward an artificial cell assembly Noireaux, V.; Libchaber, A. Proceedings of the National Academy of Sciences, Vol. 101, Issue 51 https://doi.org/10.1073/pnas.0408236101	journal	December 2004
Ultrahigh-throughput screening in drop-based microfluidics for directed evolution Agresti, J. J.; Antipov, E.; Abate, A. R. Proceedings of the National Academy of Sciences, Vol. 107, Issue 9, p. 4004-4009 https://doi.org/10.1073/pnas.0910781107	journal	February 2010
A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa -Infected Respiratory Samples Wen, Ke Yan; Cameron, Loren; Chappell, James ACS Synthetic Biology, Vol. 6, Issue 12 https://doi.org/10.1021/acssynbio.7b00219	journal	October 2017
LasR, a Transcriptional Activator of Pseudomonas aeruginosa Virulence Genes, Functions as a Multimer Kiratisin, Pattarachai; Tucker, Kenneth D.; Passador, Luciano Journal of Bacteriology, Vol. 184, Issue 17 https://doi.org/10.1128/JB.184.17.4912-4919.2002	journal	September 2002
Linear DNA for Rapid Prototyping of Synthetic Biological Circuits in an Escherichia coli Based TX-TL Cell-Free System Sun, Zachary Z.; Yeung, Enoch; Hayes, Clarmyra A. ACS Synthetic Biology, Vol. 3, Issue 6 https://doi.org/10.1021/sb400131a	journal	December 2013
Human protein factory for converting the transcriptome into an in vitro–expressed proteome Goshima, Naoki; Kawamura, Yoshifumi; Fukumoto, Akiko Nature Methods, Vol. 5, Issue 12 https://doi.org/10.1038/nmeth.1273	journal	November 2008
Identification and genetic analysis of cancer cells with PCR-activated cell sorting Eastburn, Dennis J.; Sciambi, Adam; Abate, Adam R. Nucleic Acids Research, Vol. 42, Issue 16 https://doi.org/10.1093/nar/gku606	journal	July 2014
Exploring protein fitness landscapes by directed evolution Romero, Philip A.; Arnold, Frances H. Nature Reviews Molecular Cell Biology, Vol. 10, Issue 12 https://doi.org/10.1038/nrm2805	journal	December 2009
A completely in vitro ultrahigh-throughput droplet-based microfluidic screening system for protein engineering and directed evolution Fallah-Araghi, Ali; Baret, Jean-Christophe; Ryckelynck, Michael Lab on a Chip, Vol. 12, Issue 5 https://doi.org/10.1039/c2lc21035e	journal	January 2012
Engineering specialized metabolic pathways—is there a room for enzyme improvements? Bar-Even, Arren; Salah Tawfik, Dan Current Opinion in Biotechnology, Vol. 24, Issue 2 https://doi.org/10.1016/j.copbio.2012.10.006	journal	April 2013
Regulation of virulence and antibiotic resistance by two-component regulatory systems in Pseudomonas aeruginosa Gooderham, W. James; Hancock, Robert E. W. FEMS Microbiology Reviews, Vol. 33, Issue 2 https://doi.org/10.1111/j.1574-6976.2008.00135.x	journal	March 2009
CDD: NCBI's conserved domain database Marchler-Bauer, Aron; Derbyshire, Myra K.; Gonzales, Noreen R. Nucleic Acids Research, Vol. 43, Issue D1 https://doi.org/10.1093/nar/gku1221	journal	November 2014
Biocompatible surfactants for water-in-fluorocarbon emulsions Holtze, C.; Rowat, A. C.; Agresti, J. J. Lab on a Chip, Vol. 8, Issue 10, p. 1632-1639 https://doi.org/10.1039/b806706f	journal	January 2008
An Improved Single-Chain Fab Platform for Efficient Display and Recombinant Expression Koerber, James T.; Hornsby, Michael J.; Wells, James A. Journal of Molecular Biology, Vol. 427, Issue 2 https://doi.org/10.1016/j.jmb.2014.11.017	journal	January 2015
A Paper-Based Multiplexed Transaminase Test for Low-Cost, Point-of-Care Liver Function Testing Pollock, N. R.; Rolland, J. P.; Kumar, S. Science Translational Medicine, Vol. 4, Issue 152 https://doi.org/10.1126/scitranslmed.3003981	journal	September 2012
An integrated cell-free metabolic platform for protein production and synthetic biology Jewett, Michael C.; Calhoun, Kara A.; Voloshin, Alexei Molecular Systems Biology, Vol. 4, Issue 1, Article No. 220 https://doi.org/10.1038/msb.2008.57	journal	October 2008
Paper-Based Synthetic Gene Networks Pardee, Keith; Green, Alexander A.; Ferrante, Tom Cell, Vol. 159, Issue 4 https://doi.org/10.1016/j.cell.2014.10.004	journal	November 2014
Substrate Specificity and Kinetics of Degradation of Antisense Oligonucleotides by a 3′ Exonuclease in Plasma Eder, Paul S.; DeVINE, Rene J.; Dagle, John M. Antisense Research and Development, Vol. 1, Issue 2 https://doi.org/10.1089/ard.1991.1.141	journal	January 1991
Cell-free extract based optimization of biomolecular circuits with droplet microfluidics Hori, Yutaka; Kantak, Chaitanya; Murray, Richard M. Lab on a Chip, Vol. 17, Issue 18 https://doi.org/10.1039/C7LC00552K	journal	January 2017
Bacterial Quorum-Sensing Network Architectures Ng, Wai-Leung; Bassler, Bonnie L. Annual Review of Genetics, Vol. 43, Issue 1 https://doi.org/10.1146/annurev-genet-102108-134304	journal	December 2009
Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement Isaacs, Farren J.; Carr, Peter A.; Wang, Harris H. Science, Vol. 333, Issue 6040, p. 348-353 https://doi.org/10.1126/science.1205822	journal	July 2011
Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology Chappell, James; Jensen, Kirsten; Freemont, Paul S. Nucleic Acids Research, Vol. 41, Issue 5 https://doi.org/10.1093/nar/gkt052	journal	January 2013
A paper based microfluidic device for the detection of arsenic using a gold nanosensor Nath, Peuli; Arun, Ravi Kumar; Chanda, Nripen RSC Adv., Vol. 4, Issue 103 https://doi.org/10.1039/C4RA12946F	journal	January 2014
High-throughput injection with microfluidics using picoinjectors Abate, A. R.; Hung, T.; Mary, P. Proceedings of the National Academy of Sciences, Vol. 107, Issue 45 https://doi.org/10.1073/pnas.1006888107	journal	October 2010
Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells Cole, Russell H.; Tang, Shi-Yang; Siltanen, Christian A. Proceedings of the National Academy of Sciences, Vol. 114, Issue 33 https://doi.org/10.1073/pnas.1704020114	journal	July 2017
Streamlined extract preparation for Escherichia coli -based cell-free protein synthesis by sonication or bead vortex mixing Shrestha, Prashanta; Holland, Troy Michael; Bundy, Bradley Charles BioTechniques, Vol. 53, Issue 3 https://doi.org/10.2144/0000113924	journal	September 2012
Microfluidic single-cell whole-transcriptome sequencing Streets, A. M.; Zhang, X.; Cao, C. Proceedings of the National Academy of Sciences, Vol. 111, Issue 19 https://doi.org/10.1073/pnas.1402030111	journal	April 2014
Quorum-Sensing Regulation of a Copper Toxicity System in Pseudomonas aeruginosa Thaden, J. T.; Lory, S.; Gardner, T. S. Journal of Bacteriology, Vol. 192, Issue 10 https://doi.org/10.1128/JB.01528-09	journal	March 2010
Simplifying and streamlining Escherichia coli-based cell-free protein synthesis Yang, William C.; Patel, Kedar G.; Wong, H. Edward Biotechnology Progress, Vol. 28, Issue 2 https://doi.org/10.1002/btpr.1509	journal	February 2012
Characterization of an Endonuclease IV 3′-5′ Exonuclease Activity Kerins, Sinéad M.; Collins, Ruairi; McCarthy, Tommie V. Journal of Biological Chemistry, Vol. 278, Issue 5 https://doi.org/10.1074/jbc.M210750200	journal	November 2002
3D-SIM Super Resolution Microscopy Reveals a Bead-Like Arrangement for FtsZ and the Division Machinery: Implications for Triggering Cytokinesis Strauss, Michael P.; Liew, Andrew T. F.; Turnbull, Lynne PLoS Biology, Vol. 10, Issue 9 https://doi.org/10.1371/journal.pbio.1001389	journal	September 2012
Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation Albayrak, Cem; Swartz, James R. Nucleic Acids Research, Vol. 41, Issue 11 https://doi.org/10.1093/nar/gkt226	journal	April 2013
3D-footprint: a database for the structural analysis of protein–DNA complexes Contreras-Moreira, Bruno Nucleic Acids Research, Vol. 38, Issue suppl_1 https://doi.org/10.1093/nar/gkp781	journal	September 2009
Kin cell lysis is a danger signal that activates antibacterial pathways of Pseudomonas aeruginosa LeRoux, Michele; Kirkpatrick, Robin L.; Montauti, Elena I. eLife, Vol. 4 https://doi.org/10.7554/eLife.05701	journal	February 2015
Valve-based flow focusing for drop formation Abate, Adam R.; Romanowsky, Mark B.; Agresti, Jeremy J. Applied Physics Letters, Vol. 94, Issue 2 https://doi.org/10.1063/1.3067862	journal	January 2009
Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry Harris, D. Calvin; Jewett, Michael C. Current Opinion in Biotechnology, Vol. 23, Issue 5 https://doi.org/10.1016/j.copbio.2012.02.002	journal	October 2012
Portable, On-Demand Biomolecular Manufacturing Pardee, Keith; Slomovic, Shimyn; Nguyen, Peter Q. Cell, Vol. 167, Issue 1 https://doi.org/10.1016/j.cell.2016.09.013	journal	September 2016
Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system Shin, Jonghyeon; Noireaux, Vincent Journal of Biological Engineering, Vol. 4, Issue 1 https://doi.org/10.1186/1754-1611-4-9	journal	January 2010
Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology Sun, Zachary Z.; Hayes, Clarmyra A.; Shin, Jonghyeon Journal of Visualized Experiments, Issue 79 https://doi.org/10.3791/50762	journal	January 2013
LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression Gambello, M. J.; Kaye, S.; Iglewski, B. H. Infection and Immunity, Vol. 61, Issue 4 https://doi.org/10.1128/IAI.61.4.1180-1184.1993	journal	April 1993