Name: digital pcr-based competitive index for high-throughput analysis of fitness in salmonella
Uploaded: 2019-05-13T15:00:00
Description: Watch this Scientific Journal Video about Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella at JoVE.com

Research reported in this publication was supported by the George F. Haddix President&#8217;s Faculty Research Fund and the National Institute of General Medical Science of the National Institutes of Health (NIH) under award number GM103427. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Incorporate Unique DNA Barcodes onto a Plasmid Containing the Necessary Components for Allelic Exchange
NOTE: A new plasmid, named pSKAP, with a high copy number and increased transformation efficiency compared to the existing pKD13 allelic exchange plasmid was created. This is described in steps 1.1-1.12 (Figure 1). The finalized plasmids containing unique DNA barcodes and components for allelic exchange are available through a plasmid repositor...

<ol>
	<li>Beuz&#243;n, C. R., Holden, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+mixed+infections+with+Salmonella+strains+to+study+virulence+genes+and+their+interactions+in+vivo.">Use of mixed infections with Salmonella strains to study virulence genes and their interactions in vivo.</a> Microbes and Infection. 3 (14-15), 1345-1352 (2001).</li><li>B&#228;umler, A. J., Tsolis, R. M., Valentine, P. J., Ficht, T. A., Heffron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+effect+of+mutations+in+invA+and+lpfC+on+the+ability+of+Salmonella+typhimurium+to+cause+murine+typhoid.">Synergistic effect of mutations in invA and lpfC on the ability of Salmonella typhimurium to cause murine typhoid.</a> Infection and Immunity. 65 (6), 2254-2259 (1997).</li><li>Vital, M., Hammes, F., Egli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competition+of+Escherichia+coli+O157+with+a+drinking+water+bacterial+community+at+low+nutrient+concentrations.">Competition of Escherichia coli O157 with a drinking water bacterial community at low nutrient concentrations.</a> Water Research. 46 (19), 6279-6290 (2012).</li><li>Schellenberg, J., Smoragiewicz, W., Karska-Wysocki, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+combining+immunofluorescence+and+flow+cytometry+for+improved+understanding+of+competitive+interactions+between+lactic+acid+bacteria+(LAB)+and+methicillin-resistant+S.+aureus+(MRSA)+in+mixed+culture.">A rapid method combining immunofluorescence and flow cytometry for improved understanding of competitive interactions between lactic acid bacteria (LAB) and methicillin-resistant S. aureus (MRSA) in mixed culture.</a> Journal of Microbiological Methods. 65 (1), 1-9 (2006).</li><li>Becker, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+Salmonella+metabolism+limits+possibilities+for+new+antimicrobials.">Robust Salmonella metabolism limits possibilities for new antimicrobials.</a> Nature. 440 (7082), 303 (2006).</li><li>Rang, C., Galen, J. E., Kaper, J. B., Chao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fitness+cost+of+the+green+fluorescent+protein+in+gastrointestinal+bacteria.">Fitness cost of the green fluorescent protein in gastrointestinal bacteria.</a> Canadian Journal of Microbiology. 49 (9), 531-537 (2003).</li><li>Santiviago, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+pools+of+targeted+Salmonella+deletion+mutants+identifies+novel+genes+affecting+fitness+during+competitive+infection+in+mice.">Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice.</a> PLoS Pathogens. 5 (7), e1000477 (2009).</li><li>Ahmer, B. M., Gunn, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+Salmonella+spp.+with+the+intestinal+microbiota.">Interaction of Salmonella spp. with the intestinal microbiota.</a> Frontiers in Microbiology. 2, 101 (2011).</li><li>Gallagher, L. A., Shendure, J., Manoil, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+identification+of+resistance+functions+in+Pseudomonas+aeruginosa+using+Tn-seq.">Genome-scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq.</a> MBio. 2 (1), (2011).</li><li>Van Opijnen, T., Bodi, K. L., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tn-seq:+high-throughput+parallel+sequencing+for+fitness+and+genetic+interaction+studies+in+microorganisms.">Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms.</a> Nature Methods. 6 (10), 767 (2009).</li><li>van Opijnen, T., Lazinski, D. W., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome&#8208;wide+fitness+and+genetic+interactions+determined+by+Tn&#8208;seq,+a+high&#8208;throughput+massively+parallel+sequencing+method+for+microorganisms.">Genome&#8208;wide fitness and genetic interactions determined by Tn&#8208;seq, a high&#8208;throughput massively parallel sequencing method for microorganisms.</a> Current Protocols in Microbiology. 36 (1), (2017).</li><li>Mutalik, V. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-barcoded+shotgun+expression+library+sequencing+for+high-throughput+characterization+of+functional+traits+in+bacteria.">Dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria.</a> Nature Communications. 10 (1), 308 (2019).</li><li>Yoon, H., Gros, P., Heffron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+PCR-based+competitive+index+for+high-throughput+screening+of+Salmonella+virulence+factors.">Quantitative PCR-based competitive index for high-throughput screening of Salmonella virulence factors.</a> Infection and Immunity. 79 (1), 360-368 (2011).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proceedings of the National Academy of Sciences. 97 (12), 6640-6645 (2000).</li><li>Henard, C. A., Bourret, T. J., Song, M., V&#225;zquez-Torres, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+redox+balance+by+the+stringent+response+regulatory+protein+promotes+antioxidant+defenses+of+Salmonella.">Control of redox balance by the stringent response regulatory protein promotes antioxidant defenses of Salmonella.</a> Journal of Biological Chemistry. 285 (47), 36785-36793 (2010).</li><li>Poteete, A. R., Fenton, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#955;+red-dependent+growth+and+recombination+of+phage+P22.">&#955; red-dependent growth and recombination of phage P22.</a> Virology. 134 (1), 161-167 (1984).</li><li>McCollister, B. D., Bourret, T. J., Gill, R., Jones-Carson, J., V&#225;zquez-Torres, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repression+of+SPI2+transcription+by+nitric+oxide-producing,+IFN&#947;-activated+macrophages+promotes+maturation+of+Salmonella+phagosomes.">Repression of SPI2 transcription by nitric oxide-producing, IFN&#947;-activated macrophages promotes maturation of Salmonella phagosomes.</a> Journal of Experimental Medicine. 202 (5), 625-635 (2005).</li><li>Shaw, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+enterica+serovar+Typhimurium+has+three+transketolase+enzymes+contributing+to+the+pentose+phosphate+pathway.">Salmonella enterica serovar Typhimurium has three transketolase enzymes contributing to the pentose phosphate pathway.</a> Journal of Biological Chemistry. 293 (29), 11271-11282 (2018).</li><li>Freter, R., O'Brien, P., Macsai, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+chemotaxis+in+the+association+of+motile+bacteria+with+intestinal+mucosa:+in+vivo+studies.">Role of chemotaxis in the association of motile bacteria with intestinal mucosa: in vivo studies.</a> Infection and immunity. 34 (1), 234-240 (1981).</li><li>Taylor, R. K., Miller, V. L., Furlong, D. B., Mekalanos, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+phoA+gene+fusions+to+identify+a+pilus+colonization+factor+coordinately+regulated+with+cholera+toxin.">Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin.</a> Proceedings of the National Academy of Sciences. 84 (9), 2833-2837 (1987).</li><li>Nogva, H. K., Dromtorp, S., Nissen, H., Rudi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethidium+monoazide+for+DNA-based+differentiation+of+viable+and+dead+bacteria+by+5'-nuclease+PCR.">Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR.</a> Biotechniques. 34 (4), 804-813 (2003).</li><li>Nocker, A., Camper, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+approaches+toward+preferential+detection+of+viable+cells+using+nucleic+acid+amplification+techniques.">Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques.</a> FEMS Microbiology Letters. 291 (2), 137-142 (2009).</li><li>Nocker, A., Cheung, C. -. Y., Camper, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+propidium+monoazide+with+ethidium+monoazide+for+differentiation+of+live+vs.+dead+bacteria+by+selective+removal+of+DNA+from+dead+cells.">Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells.</a> Journal of Microbiological Methods. 67 (2), 310-320 (2006).</li><li>Nocker, A., Mazza, A., Masson, L., Camper, A. K., Brousseau, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+detection+of+live+bacteria+combining+propidium+monoazide+sample+treatment+with+microarray+technology.">Selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology.</a> Journal of Microbiological Methods. 76 (3), 253-261 (2009).</li><li>Nocker, A., Sossa, K. E., Camper, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+monitoring+of+disinfection+efficacy+using+propidium+monoazide+in+combination+with+quantitative+PCR.">Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR.</a> Journal of Microbiological Methods. 70 (2), 252-260 (2007).</li><li>Nocker, A., Sossa-Fernandez, P., Burr, M. D., Camper, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+propidium+monoazide+for+live/dead+distinction+in+microbial+ecology.">Use of propidium monoazide for live/dead distinction in microbial ecology.</a> Applied and Environmental Microbiology. 73 (16), 5111-5117 (2007).</li><li>Price-Carter, M., Tingey, J., Bobik, T. A., Roth, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Alternative+Electron+Acceptor+Tetrathionate+Supports+B12-Dependent+Anaerobic+Growth+ofSalmonella+enterica+Serovar+Typhimurium+on+Ethanolamine+or+1,+2-Propanediol.">The Alternative Electron Acceptor Tetrathionate Supports B12-Dependent Anaerobic Growth ofSalmonella enterica Serovar Typhimurium on Ethanolamine or 1, 2-Propanediol.</a> Journal of Bacteriology. 183 (8), 2463-2475 (2001).</li><li>Taylor, R. G., Walker, D. C., McInnes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E.+coli+host+strains+significantly+affect+the+quality+of+small+scale+plasmid+DNA+preparations+used+for+sequencing.">E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing.</a> Nucleic Acids Research. 21 (7), 1677 (1993).</li><li>Cherepanov, P. P., Wackernagel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+disruption+in+Escherichia+coli:+Tc+R+and+Km+R+cassettes+with+the+option+of+Flp-catalyzed+excision+of+the+antibiotic-resistance+determinant.">Gene disruption in Escherichia coli: Tc R and Km R cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant.</a> Gene. 158 (1), 9-14 (1995).</li></ol>

The ability to accurately quantify microorganisms is of paramount importance to microbiology research, and the ability to enumerate unique strains from an initial mixed population has proved to be an invaluable tool for assessing fitness and virulence traits in bacteria. However, the techniques for accomplishing this have not progressed in pace with modern developments in molecular biology. The technology to easily modify the chromosomes of many bacteria, including S. Typhimurium, has been available for nearly t...

Assessing fitness and virulence of pathogenic organisms is a fundamental aspect of microbiology research. It enables comparisons to be made between strains or between mutated organisms, which allows researchers to determine the importance of certain genes under specific conditions. Traditionally, virulence assessment utilizes an animal model of infection using different bacterial strains and observing the outcome of the infected animal (e.g. Infectious Dose50, Lethal Dose50, time to death, symptom severity, lack of symptoms, etc.). This procedure provides valuable descriptions of virulence, but it requires strains to cause considerable differences in outcomes in order to detect variations from wild-type. Furthermore, results are semi-quantitative because while disease progression and symptom severity can be subjectively quantified over time, interpretation of virulence compared to wild-type is more qualitative (i.e. more, less, or equally virulent). A common alternative to performing animal infectivity assays is to generate competitive indices (CIs), values that directly compare fitness or virulence of a strain to a wild-type counterpart in a mixed infection1. This technique has numerous advantages over a traditional animal model of infection by standardizing virulence to a wild-type strain and determining a quantifiable value to reflect the degree of attenuation. This technique can also be adapted to analyze gene interactions in bacteria by determining a canceled out competitive index (COI)2. Calculating a COI for a group of mutated organisms allows researchers to determine whether two genes independently contribute to pathogenesis or if they are involved in the same virulence pathway and dependent on each other. Additionally, calculating a CI requires enumeration of bacteria which can provide valuable insights into the pathogenesis of organisms. CIs and COIs also allow researchers to asses avirulent strains that do not cause clinical disease but still have differences in fitness. This technique is limited by the use of traditional antibiotic resistance markers to identify strains, thereby limiting the number of input strains to only one or two at a time. Because of this limitation, large numbers of experimental groups and replicates are required, which in addition to adding to labor and material costs, also increases opportunities for variability in experimental conditions and inaccurate results. (For a thorough review of the benefits and applications of using mixed infections to study virulence, fitness, and gene interactions, see C.R. Beuz&#243;n and D.W. Holden 1)
Attempts have been made to overcome this limitation, such as the use of fluorescently-labeled cells quantified via flow cytometry3,4,5. This technique quantifies cells using either 1) labeled antibodies to phenotypic markers or 2) endogenously produced fluorescent proteins. The use of labeled antibodies has a limit of detection of 1,000 cells/mL, and therefore requires a high number of cells to analyze3. Cells expressing fluorescent proteins have an altered physiology and are susceptible to fitness changes resulting from high protein expression6. Both methods are limited by the number of fluorescent markers detectable using flow cytometry. An advancement in molecular quantification was achieved through the development of a microarray technique that detected attenuation in 120 strains from an initial mixed infection of over 1,000 strains in a murine model7. This technique utilized a microarray analysis of RNA from mutated strains, which lead to considerable variability in the outcome.&#160; Nevertheless, it established that large pools of mixed infections can be a useful tool and that by utilizing sensitive detection techniques, differences in bacterial virulence can be identified. With the development of the next generation sequencing, Tn-seq expanded the utility of transposon mutations, enabling a powerful method for quantifying bacteria that were randomly mutated8,9,10,11. An alternative protocol was recently developed that eliminates the need for transposons and instead uses DNA barcodes to more easily identify and track genomic changes and their impact on fitness12. This technology is a major advancement, but the insertion of the genomic barcodes is still a random process. To overcome the randomness of previous experiments, Yoon et al. developed a method to calculate the CIs of Salmonella strains using unique DNA barcodes inserted at precise locations on the chromosomes of bacteria13. Unique barcoded strains were detected using a qPCR-based method with SYBR green and primers specific to each unique barcode. The technique was limited by constraints imposed by qPCR, including differences in primer efficiencies and low sensitivity, evidenced by the need for nested-PCR prior to qPCR. Nevertheless, this approach demonstrated that targeted genomic modifications could be exploited for detecting and potentially quantifying pools of multiple bacterial strains.
In the following protocol, we describe a novel methodology to perform bacterial competition experiments with large pools of mixed inocula followed by accurate quantification using a highly sensitive digital PCR technique. The protocol involves genetically-labeling bacterial strains with a unique DNA barcode inserted on an innocuous region of the chromosome. This modification allows strains to be quickly and accurately quantified using modern molecular technology instead of traditional serial dilutions, replica plating, and counting colony forming units that rely on phenotypic markers (i.e. antibiotic resistance). The modifications allow for simultaneous assessment of many strains in a single pooled inoculum, substantially reducing the possibility of experimental variability because all strains are exposed to the exact same conditions. Furthermore, while this technique was developed in Salmonella enterica serovar Typhimurium, it is highly adaptable to any genetically malleable organism and nearly any experimental design where accurate bacterial counts are required, providing a new tool to increase accuracy and throughput in microbiology laboratories without the constraints imposed by previous methods.

<table>
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>22600028</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>16 mL culture tubes</td>
			<td>MidSci</td>
			<td>8599</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>5-200 &#956;L pipette tips</td>
			<td>RAININ</td>
			<td>30389241</td>
			<td>Procure alternative tip brands with caution based on manufacturing quality</td>
		</tr>
		<tr>
			<td>5-50 &#956;L multichannel pipette</td>
			<td>RAININ</td>
			<td>17013804</td>
			<td>Use alternative multichannel pipettes with caution</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>ThermoFisher Scientific</td>
			<td>BP160-500</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>BLAST Analysis</td>
			<td>NCBI</td>
			<td>N/A</td>
			<td>https://blast.ncbi.nlm.nih.gov/Blast.cgi</td>
		</tr>
		<tr>
			<td>C1000 Touch Thermocycler with 96-Deep Well Reaction Module</td>
			<td>Bio Rad</td>
			<td>1851197</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>Chemically competent DH5&#945;</td>
			<td>Invitrogen</td>
			<td>18258012</td>
			<td>Procure from any manufacturer or prepare yourself</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>ThermoFisher Scientific</td>
			<td>BP904-100</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Cytation5 Microplate reader</td>
			<td>BioTek</td>
			<td>CYT5MF</td>
			<td>Procure from any manufacturer, use any system capable of accurately quantifying DNA</td>
		</tr>
		<tr>
			<td>Data Analysis Software (QuantaSoft and QuantaSoft Data Analysis Pro)</td>
			<td>Bio Rad</td>
			<td>N/A</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>ddPCR 96-Well Plates</td>
			<td>Bio Rad</td>
			<td>12001925</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>ddPCR Droplet Reader Oil</td>
			<td>Bio Rad</td>
			<td>1863004</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>ddPCR Supermix for Probes (No dUTP)</td>
			<td>Bio Rad</td>
			<td>1863024</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>DG8 Cartridges for QX200/QX100 Droplet Generator</td>
			<td>Bio Rad</td>
			<td>1864008</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>DG8 Gaskets for QX200/QX100 Droplet Generator</td>
			<td>Bio Rad</td>
			<td>1863009</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>Droplet Generation Oil for Probes</td>
			<td>Bio Rad</td>
			<td>1863005</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>ThermoFisher Scientific</td>
			<td>BP906-5</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Luria-Bertani agar</td>
			<td>ThermoFisher Scientific</td>
			<td>BP1425-2</td>
			<td>Procure from any manufacturer or make it yourself from agar, tryptone, yeast digest, and NaCl</td>
		</tr>
		<tr>
			<td>Luria-Bertani broth</td>
			<td>ThermoFisher Scientific</td>
			<td>BP1426-2</td>
			<td>Procure from any manufacturer or make it yourself from tryptone, yeast digest, and NaCl</td>
		</tr>
		<tr>
			<td>PCR Plate Heat Seal, foil, pierceable</td>
			<td>Bio Rad</td>
			<td>1814040</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>PCR Tubes</td>
			<td>Eppendorf</td>
			<td>951010022</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>ThermoFisher Scientific</td>
			<td>FB0875712</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>pPCR Script Cam SK+</td>
			<td>Stratagene/Agilent</td>
			<td>211192</td>
			<td>No longer available commercially</td>
		</tr>
		<tr>
			<td>Primer/Probe Design</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>https://www.idtdna.com/Primerquest/Home/Index</td>
		</tr>
		<tr>
			<td>pSKAP and pSKAP_Barcodes</td>
			<td>Addgene</td>
			<td>Plasmid numbers 122702-122726</td>
			<td>www.addgene.org</td>
		</tr>
		<tr>
			<td>PX1 PCR Plate Sealer</td>
			<td>Bio Rad</td>
			<td>1814000</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>QX200 Droplet Generator</td>
			<td>Bio Rad</td>
			<td>1864002</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader</td>
			<td>Bio Rad</td>
			<td>1864003</td>
			<td>Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.</td>
		</tr>
		<tr>
			<td>S. Typhimurium strain ATCC 14028s</td>
			<td>ATCC</td>
			<td>ATCC 14028s</td>
			<td>www.atcc.org</td>
		</tr>
		<tr>
			<td>Take3 Micro-Volume Plate</td>
			<td>BioTek</td>
			<td>TAKE3</td>
			<td>Procure from any manufacturer, use any system capable of accurately quantifying DNA</td>
		</tr>
		<tr>
			<td>Thermo Scientific FastDigest BamHI</td>
			<td>ThermoFisher Scientific</td>
			<td>FERFD0054</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermo Scientific FastDigest DpnI</td>
			<td>ThermoFisher Scientific</td>
			<td>FERFD1704</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermo Scientific FastDigest HindIII</td>
			<td>ThermoFisher Scientific</td>
			<td>FERFD0504</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermo Scientific GeneJet Gel Extraction and DNA Cleanup Micro Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>FERK0832</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermo Scientific GeneJet Miniprep Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>FERK0503</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermo Scientific Phusion High-Fidelity DNA Polymerase</td>
			<td>ThermoFisher Scientific</td>
			<td>F534L</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermo Scientific T4 DNA Ligase</td>
			<td>ThermoFisher Scientific</td>
			<td>FEREL0011</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Bio Rad</td>
			<td>1861096</td>
			<td>Procure from any manufacturer</td>
		</tr>
		<tr>
			<td>UVP Visi-Blue Transilluminator</td>
			<td>ThermoFisher Scientific</td>
			<td>UV95043301</td>
			<td>Or other transiluminator that allows visualization of DNA</td>
		</tr>
		<tr>
			<td>Water, Molecular Biology Grade</td>
			<td>ThermoFisher Scientific</td>
			<td>BP28191</td>
			<td>Procure from any manufacturer</td>
		</tr>
	</tbody>
</table>

digital pcr-based competitive index for high-throughput analysis of fitness in salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

The use of this methodology requires that appropriate control reactions are performed to validate the sensitivity and specificity of each probe used to identify target DNA. In this representative experiment, we validated eight unique DNA barcodes with the eight corresponding probes for identification. All eight probes had a low rate of false positives in both NTC and negative control reactions (Table 3), highlighting their specificity even among highly similar DNA sequenc...

Watch this Scientific Journal Video about Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella at JoVE.com

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

This technique enables accurate and precise quantification of microorganisms from a mixed population of highly similar strains using rapid and high throughput molecular-based techniques. Ultimately, in enabling comparisons of fitness to be made between strains. The ability to simultaneously assess many strains in a single pool substantially reduces well-to-well or organism-to-organism variability because all strains are exposed to precisely the same conditions.The adaptability of this technique allows it to be used for nearly any genetically malleable organism and nearly any experimental design that requires accurate quantification of microbes. Begin this procedure with engineering of genetic markers onto bacterial chromosomes, as described in the text protocol. Create a pool of genomic DNA that contains every barcode except for one.Use this pool as the diluent to perform a dilution series with genomic DNA containing the single remaining barcode. Prepare reactions for digital PCR in duplicate according to the manufacturer's recommendations for using a digital PCR supermix designed for probe-based chemistry. Use the pooled genomic DNA as template DNA.Also, add the 20 X primer probe Master Mix as described in the text protocol. Prepare replicate control reactions for digital PCR according to the manufacturer's recommendations. Control reactions for each probe mix must consist of No template controls, negative controls, and positive controls.Repeat these steps to create digital PCR reactions for each barcoded genomic DNA sample. Generate droplets for each reaction condition using a droplet generator, according to the manufacturer's instructions. Transfer newly created droplets into the appropriate 96 well plate.Use 200 microliter pipette tips on a 5 through 50 microliter multi-channeled pipette. After all the droplets have been generated and transferred, seal the plate with a foil plate sealer. Use the manufacturer recommended thermal cycler to perform the cycling conditions.While thermocycling is being performed, program the data analysis software with the plate setup information, such as sample name, experiment type, and Supermix used. Also include Target 1 name and Target 1 type, as well as Target 2 name and Target 2 type. After thermocycling is complete, transfer the completed reactions to the droplet reader and start the reading process, according to the manufacturer's instructions.When all wells have been read and the run is complete, open the QLP data file using the data analysis software. Select all wells that utilize the same primer probe Mastermix. On the Droplets tab, examine the number of droplets analyzed in each well, including both positive and negative droplets.Exclude from analysis any well that is fewer than 10, 000 total droplets. Move to the 1D Amplitude tab and examine the amplitudes of positive and negative droplets. Ensure they comprise 2 distinct populations.Within the software, use the thresholding feature to make a cutoff between positive and negative droplets for each probe that was utilized. Once appropriate thresholds have been applied to all wells, the software will calculate the number of DNA copies in each reaction. Export data to a spreadsheet to facilitate further analysis.Using these values, calculate the initial copy number of each unique genomic barcode in the sample. Determine the mean false positive rate from the negative control reactions and subtract this value from the values obtained in experimental reactions. Multiply the values as necessary, based on the dilutions that were performed when setting up each experiment.Now, determine the relative fitness of an organism by calculating the competitive index or canceled out competitive index from digital PCR-based quantification of the barcoded strain. Perform this step for each barcoded strain at all time points. Genomic DNA containing the AA barcode was diluted in a background of all other barcoded genomic DNA.Channel 1 represents the FAM probe for the AA barcode, while Channel 2 represents the HEX probe for the AO barcode. Results of each probe are presented as both individual droplet fluorescent amplitude and a histogram representing the frequency of fluorescent intensity of all droplets in the selected wells. For each condition, positive and negative droplets should form 2 distinct populations.The populations should be separated using the thresholds feature to define positive and negative droplets. Threshold values will vary depending on the probes that were used, but all wells that utilize the same probe mix should have identical thresholds. In the case of AA that was diluted, the histogram appears to only depict the single population because positive droplets are substantially outnumbered by negative droplets.However, 2 distinct populations are still visible by examining droplet fluorescent amplitude in the left panels. As the AA barcoded genomic DNA was diluted, there was a decrease in positive droplets, while the number of positive AO droplets remains constant. Once appropriate thresholds have been applied to all wells the software will calculate the number of DNA copies in each reaction.If you routinely perform PCR as part of your research, you can perform this technique to assess the fitness of your model organism. This procedure would normally be used to determine end results of upstream experiments. Its true utility is to promote ease and versatility for any experiments that rely on bacterial quantification.We are using this technique to assess salmonella fitness in a murine model of infection. Additionally, this technique is being adapted for use with other pathogenic microorganisms in our laboratory.

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Research

JoVE Journal

Genetics

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures &#8212; temperature and osmolarity &#8212; across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...