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 authors have nothing to disclose.

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

9.5K Views.  Creighton University School of Medicine. 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 ...