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

Summary

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.

Abstract

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.

Introduction

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. Beuzón and D.W. Holden ¹)

Attempts have been made to overcome this limitation, such as the use of fluorescently-labeled cells quantified via flow cytometry^3,4,5. 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.  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 mutated^8,9,10,11. 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.

Protocol

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 repository (Table of Materials).

1. Using a commercial plasmid miniprep kit, purify pKD13¹⁴ and pPCR Script Cam SK⁺ from overnight bacterial cultures grown in Luria-Bertani (LB) broth supplemented with 50 μg/mL kanamycin or 25 μg/mL chloramphenicol (for pKD13 and pPCR Script Cam SK+, respectively) (Table 1).
2. Perform restriction digestions on both plasmids using commercial restriction enzymes HindIII and BamHI according to the manufacturer’s specifications.
3. Remove restriction enzymes and excised DNA from the pPCR Script Cam SK⁺ reaction and purify the 3,370-base pair (bp) plasmid backbone using a commercially available DNA cleanup kit according to the manufacturer’s specifications.
4. Separate the fragments from the pKD13 restriction digestion on a 1% agarose gel using an electrophoresis chamber.
5. Visualize bands using a blue light transilluminator and excise the 1,333 bp fragment from the gel (Figure 1C).
   NOTE: This fragment contains the FRT-flanked kanamycin resistance gene required for chromosomal allelic replacement.
6. Purify the excised DNA from step 1.5 using a commercial gel extraction kit.
7. To create pSKAP, ligate the purified fragment from pKD13 (from step 1.6) into pPCR Script Cam SK⁺ (from step 1.3) using a commercial T4 DNA ligase according to the manufacturer’s specifications.
8. Transform chemically competent DH5α cells with the ligated pSKAP plasmid following the manufacturer’s protocol (Table 1).
9. Spread transformants onto LB agar plates supplemented with 50 μg/mL kanamycin and incubate at 37 °C overnight.
10. Pick a colony from the plate and streak it on a new LB agar plate supplemented with 50 μg/mL kanamycin and incubate at 37 °C overnight. Pick a colony from this plate and use to inoculate LB broth supplemented with 50 μg/mL kanamycin. Incubate the culture overnight at 37 °C with constant agitation.
11. Use a commercial plasmid miniprep kit to purify pSKAP from the overnight bacterial culture.
12. Perform a diagnostic restriction digestion of the plasmid from step 1.11 using Hind III and Bam HI according to manufacturer’s specifications. Visualize fragments on a 1% agarose gel as in steps 1.4-1.5.
    NOTE: The pSKAP total size should be 4,703 bp. Fragments after step 1.12 should be 3,370 and 1,333 bp.
13. Design PCR primers for insertional site-directed mutagenesis (SDM) (Table 2 and S1) in such a way as to insert a unique 25-basepair DNA sequence at position 725 of pSKAP (Figure 2).
    NOTE: The barcode DNA is inserted into the plasmid just outside of the FRT-flanked kanamycin resistance gene, so the barcode is not lost during subsequent removal of the kanamycin resistance cassette. If generating new barcode sequences, use online tools to ensure that the fluorescently-labeled target-specific PCR probes (hereafter referred to simply as “probes”) will efficiently bind to the new sequence. Insertion sequences designed to-date, along with the necessary primers to create them, are provided in Tables 2 and S1.
14. Prepare SDM reactions using a commercial high-fidelity DNA polymerase, the desired primer pairs (Table 2), and pSKAP template. Set the thermocycler to perform the following: 1) 98 °C for 30 s, 2) 98 °C for 10 s, 56 °C for 15 s, 72 °C for 2 min, 3) repeat steps 2, 24 times, 4) 72 °C for 5 min, 5) hold at 4 °C.
15. After completion of PCR, deplete pSKAP template by adding the restriction enzyme Dpn I to the reaction. Incubate at 37 °C for 20 min.
    NOTE: Products from PCR should be visualized on an agarose gel to verify the size and purity of the product. A control Dpn I digestion consisting of the unmodified pSKAP template can be performed and used in subsequent steps to ensure template DNA is completely digested.
16. Use 5 μL of the product from step 1.15 to transform 100 μL of commercial chemically competent DH5α cells according to the manufacturer’s recommendations.
17. Spread transformants onto LB agar plates supplemented with 25 μg/mL chloramphenicol and incubate at 37 °C overnight.
18. Select a colony (or colonies) from overnight plates and streak onto individual LB agar plates supplemented with 25 μg/mL chloramphenicol and incubate at 37 °C overnight. Select a colony from overnight plates and use to inoculate 5 mL of LB broth supplemented with 25 μg/mL chloramphenicol. Incubate culture(s) overnight at 37 °C with constant agitation.
19. Use a commercial plasmid miniprep kit to purify plasmids from the overnight culture(s).
20. Sanger sequence purified plasmids using the M13 Forward sequencing primer (Table 2). Compare mutated region to the original plasmid and assess for SDM insertional accuracy.
21. After confirming the barcode insertion and accuracy, assign each barcode and plasmid a name.
    NOTE: Barcodes generated to-date have been assigned a two-letter designation: AA, AB, AC, …, BA, BB, BC, etc. Barcoded plasmids are denoted as pSKAP_AA, pSKAP_AB, pSKAP_AC, …, pSKAP_BA, pSKAP_BB, pSKAP_BC, etc.
22. Repeat steps 1.14-1.21 to generate the desired number of DNA barcodes.

2. Introduce DNA Barcode onto the Chromosome of S. Typhimurium

NOTE: Insertion of DNA barcodes onto the S. Typhimurium chromosome is achieved by using an allelic exchange method described by Datsenko and Wanner¹⁴ that has been modified for use in S. Typhimurium.

1. Determine the locus on the S. Typhimurium genome at which to insert the DNA barcode (Figure 3).
   NOTE: Select a large, intergenic region of the chromosome. Avoid regions that produce non-coding RNA. This study utilized a locus downstream of put P between residues 1,213,840 and 1,213,861 (determined from genome assembly GCA_000022165.1). This region has previously been genetically manipulated for in trans complementation of genes¹⁵. Alternatively, a DNA barcode could be introduced while simultaneously disrupting a gene of interest. Doing so would require minimal alterations to this protocol and streamline mutant creation.
2. Design PCR primers to amplify the unique barcode and FRT-flanked kanamycin resistance gene from the desired pSKAP barcode-containing plasmid from step 1.21 (Table 2). Add 40-nucleotide extensions that are homologous to the region selected in step 2.1 to the 5’ end of each primer (Table 2).
3. Perform the amplification using a commercial high-fidelity polymerase, the primers from step 2.2, and the desired pSKAP barcode-containing plasmid as template. Set the thermocycler to perform the following: 1) 98 °C for 30 s, 2) 98 °C for 10 s, 3) 56 °C for 15 s, 4) 72 °C for 60 s, repeat steps 2-4 29 times, 5) 72 °C for 5 min, 6) hold at 4 °C.
4. After completion of PCR, deplete template DNA using DpnI as described in step 1.15. Purify and concentrate the DNA using a commercial DNA cleanup kit according to the manufacturer’s specifications.
5. Refer to the previously published protocol for generating mutants in S. Typhimurium strain 14028s from PCR products^14,16,17,18.
   NOTE: It is not essential that the kanamycin resistance gene is excised from the chromosome. However, excision of the gene is minimally disruptive to the bacterial chromosome as it results in a 129 bp scar that would leave potential downstream genes in-frame. It is recommended that the strain containing the kanamycin resistance gene is retained as it can be used to move barcodes between strains via P22-mediated transduction.
6. Repeat steps 2.3 – 2.5 to create the desired strains with the appropriate barcodes.
   NOTE: Barcodes can be introduced into wild-type S. Typhimurium that can then be subjected to further genetic manipulation, or barcodes can be introduced into strains that have been previously genetically altered.

3. Bacterial Growth Conditions and In Vitro Competition Assays

1. From bacteria stock, streak desired S. Typhimurium strains that each harbor a unique DNA barcode onto LB agar plates. Incubate plates overnight at 37 °C.
2. Select a single colony from each strain and inoculate 5 mL of LB broth. Incubate for 20 h at 37 °C with constant agitation.
   NOTE: Using the overnight culture, proceed to step 3.5 to collect pure genomic DNA (gDNA) from each barcoded strain. This is necessary for subsequent validation and control experiments in section 6.
3. For each competition assay, transfer an equal volume of each overnight culture into an appropriately sized sterile tube. Thoroughly mix strains together by vortexing vigorously for at least 5 s.
   NOTE: The volume of each overnight culture to transfer should be sufficient for each condition and replicate, as well as for isolating gDNA to quantify input. While it is not entirely necessary to measure optical densities of cultures because the absolute number of input microorganisms will be quantified using digital PCR, the number of input bacteria for each strain should be approximately equal to avoid bottlenecks or unequal competition early in the experiment. Representative competition assays in this protocol compared growth rates of 8 strains simultaneously. Additional or fewer strains may be necessary for individual experimental designs.
4. Transfer 100 μL of the mixed inoculum into 4.9 mL sterile LB broth. Incubate at 37 °C for the desired time or to a desired optical density.
5. Harvest 500 μL of the inoculum by centrifugation at >12,000 x g for 1 min. Remove and discard the supernatant. Proceed immediately to section 4 with the cells.
6. At desired time points, remove 500 μL aliquots of culture and harvest cells by centrifugation at >12,000 x g for 1 min. Remove and discard supernatant.
   NOTE: If collecting aliquots at multiple timepoints, freeze pellets at -20 °C or immediately proceed to step 4.1 after each collection.

4. Collecting and Quantifying gDNA from S. Typhimurium (from Steps 3.5 and 3.6)

1. Harvest gDNA from cells using a commercial gDNA purification kit. If available, perform the optional RNA depletion step.
   NOTE: RNA depletion is not necessary; however, the presence of RNA will artificially increase the DNA concentration, leading to aberrant calculations in subsequent steps. If using a commercial gDNA purification kit, follow the manufacturer’s recommendations to ensure that the column is not overloaded with DNA. There is no minimum DNA concentration required if the sample is quantifiable in subsequent steps.
2. Use a spectrophotometer to quantify DNA in each sample.
   NOTE: DNA can be quantified using any reliable method.
3. Calculate gDNA copy number based on the bacterial genome size using the following equation where: X is the amount of DNA in ng and N is the length of a double-stranded DNA molecule (the genome size).
   
   NOTE: 660 g/mole is used as the average mass of 1 DNA bp. Small variations may exist depending on the organism’s nucleotide composition. Numerous calculators are available online to perform the calculation.

5. Design Primers and Probes for Quantitative Detection of DNA Barcodes via dDigital PCR

1. Design primers to amplify the barcoded region of the S. Typhimurium chromosome downstream of putP (Figure 3C and Table 2).
   NOTE: Primer and probe designs can be facilitated by numerous online programs (Table of Materials). If barcodes are all inserted at the same loci, a single set of amplification primers is universal for all barcodes.
2. Design 6-carboxyfluorescein (FAM)-based and/or hexachlorofluorescein (HEX)-based probes specific to each barcode (Table 2 and S1).
   NOTE: The droplet reader used in this experiment is capable of detecting both FAM- and HEX-based probes simultaneously in a multiplex reaction. Design ½ of the probes to utilize FAM and ½ to utilize HEX. This is not a necessary step but will reduce the reagent use and experimental costs if implemented.
3. Make 20x primer-probe master mixes containing 1) 20 mM of each forward and reverse amplification primer, 2) 10 mM of a single FAM probe, and 3) 10 mM of a single HEX probe (if multiplexing).

6. Validate the Sensitivity and Specificity of Each Pprimer-probe Set for Each Genomic Barcode Using Digital PCR

NOTE: This protocol uses validating eight unique barcodes with eight unique probes as an example. The number of barcodes utilized can be increased or decreased to accommodate various experimental designs.

1. Create a pool of gDNA that contains every barcode except for one. Use this pool as the diluent to perform a dilution series with gDNA containing the single remaining barcode (sample dilution scheme is provided in Figure 4).
   NOTE: Using pooled gDNA as a diluent ensures a consistent background while ascertaining sensitivity. Using the copy numbers determined in step 4.3, dilute gDNA to a copy number within the recommended digital PCR range (1-100,000 copies per 20 μL reaction). Keep in mind that the range is set for each unique target (barcode), not the total gDNA.
2. 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 mixtures from step 6.1 as template DNA. Use the 20x primer-probe master mix from step 5.3 that contains the probe for the diluted barcode in step 6.1.
3. Prepare replicate control reactions for digital PCR according to the manufacturer’s recommendations for using a digital PCR supermix designed for probe-based chemistry. Control reactions for each probe mix must consist of 1) no template controls (NTCs), 2) negative controls, and 3) positive controls.
   NOTE: The minimum number of replicate control reactions is two. This example protocol uses four NTCs, six negative controls, and six positive controls for each barcode. Negative controls should contain gDNA with each barcode except for the barcode corresponding to the probe being tested. This will validate the specificity of each probe.
4. Repeat steps 6.1-6.3 to create digital PCR reactions for each barcoded gDNA sample. A sample plate arrangement is presented in Figure 5.
   NOTE: This and subsequent steps are described based on a specific digital PCR platform that utilizes droplets and flow-based technology. Alternative digital PCR platforms that utilize chip-based technology can easily be substituted with slight modifications to this protocol. Step 6.4 may require more than one 96-well plate to validate all primer sets. In contrast to qPCR, separate plates analyzed by digital PCR can be readily compared without the need for standardized reference wells between plates.
5. Generate droplets for each reaction condition using a droplet generator according to the manufacturer’s instructions.
6. Transfer newly created droplets into the appropriate 96-well plate. Use 200 μL pipette tips on a 5-50 μL multichannel pipette.
   NOTE: When pipetting droplets, pipette slowly and smoothly! The digital PCR equipment manufacturer recommends using only pipettes and pipette tips from a particular manufacturer (e.g., Ranin). These pipette tips have a smooth opening with no microscopic plastic fragments that can destroy droplets or damage the microfluidics of the droplet reader. Numerous brands of tips were examined and observed to have a spectrum of manufacturing quality. Equivalent results have been achieved using pipette tip alternatives; however, caution should be used when deviating from the manufacturer’s recommendations.
7. After all the droplets have been generated and transferred, seal the plate with a foil plate sealer.
8. Use the manufacturer-recommended thermocycler to perform the following cycling conditions: 1) 94 °C for 10 min; 2) 94 °C for 1 min, ramp rate set at 1 °C/s; 3) 55 °C for 2 min, ramp rate set at 1 °C/s; 4) repeat steps 2 and 3 49 times; 5) 98 °C for 10 min; 6) hold at 4 °C up to 24 h.
   NOTE: Thermal transfer in a droplet reaction is not the same as standard PCR. Reaction conditions may require modification.
9. While thermocycling is being performed, program the data analysis software with the plate setup information such as sample name, experiment type (absolute quantification), supermix used, target 1 name (FAM barcode name), target 1 type (NTC, positive control, negative control, or unknown), target 2 name (HEX barcode name), target 2 type (blank, positive control, negative control, or unknown). The final plate setup information is shown in Figure 5.
10. After thermocycling is complete, transfer the completed reactions to the droplet reader and start the reading process according to the manufacturer’s instructions.

7. Quantify the Number of Bacteria in a Competitive Index Experiment

1. Dilute gDNA isolated and quantified from section 4 to an appropriate concentration as described above.
2. Prepare reactions for digital PCR according to the manufacturer’s recommendations for using the appropriate supermix. Use DNA from step 7.1 as the template. Use one 20x primer-probe master mix that contains the probe (or probes if detecting both FAM and HEX) for possible barcodes present in the experiment.
3. Prepare additional digital PCR reactions as in step 7.2 using different 20x primer-probe master mixes until all barcodes utilized in the experimental design can be detected.
4. Include controls for each condition as described in 6.3. This includes 1) no template controls (NTCs), 2) negative controls, and 3) positive controls.
5. Continue with the protocol as described in steps 6.5-6.10.

8. Analyze Digital PCR Data and Calculate Absolute Copy Numbers

1. When all wells have been read and the run is complete, open the .qlp data file using the data analysis software.
   NOTE: The file types and data analysis procedures described here are specific to one digital PCR manufacturer. If using alternative digital PCR platforms, file types and data analysis procedures will be specific to the platform used and should be performed according to the manufacturer’s recommended specifications.
2. Select all wells that utilize the same primer-probe master mix.
3. On the Droplets tab, examine the number of droplets analyzed in each well (both positive and negative droplets). Exclude from analysis any well that has fewer than 10,000 total droplets.
4. Move to the 1D Amplitude tab and examine the amplitudes of positive and negative droplets. Ensure they comprise two distinct populations.
5. Within the software, use the thresholding feature to make a cutoff between positive and negative droplets for each probe that was utilized (Figure 6).
   NOTE: All wells that use the same primer-probe master mix from step 5.3 should have the same thresholds.
6. 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.
   NOTE: The data analysis software uses the number of positive and negative droplets that are fit to a Poisson distribution to determine the copy number.
7. Using the values from step 8.6, 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 values as necessary based on the dilutions that were performed when setting up each experiment (Table 3).

9. Determine Relative Fitness of an Organism by Calculating the CI or COI from Digital PCR-based Quantification of Barcoded Strains

1. Calculate the CI of a barcoded strain using the following formulas where: A_Output is the absolute quantification of the barcoded strain at a given timepoint, WT_Output is the absolute quantification of barcoded wild-type bacteria at the same timepoint, A_Input is the absolute quantification of the input inoculum of the barcoded strain, WT_Input is the absolute quantification of the input inoculum of barcoded wild-type bacteria, X_Output is the summation of all strains at the same timepoint, X_Input is the summation of the total input inoculum of all barcoded strains.

NOTE: See the discussion for advantages, disadvantages, and the most appropriate use of each formula.

2. Repeat step 9.1 for each barcoded strain at all timepoints.
3. If applicable, calculate the COI using the following formulas where: A_Output is the absolute quantification of a barcoded strain with mutated gene A at a given timepoint, AB_Output is the absolute quantification of a barcoded strain with mutated genes A and B at the same timepoint, A_Input is the absolute quantification of the input inoculum of the barcoded strain with mutated gene A, AB_Input is the absolute quantification of the input inoculum of barcoded strain with mutated genes A and B, B_Output is the absolute quantification of a barcoded strain with mutated gene B at a given timepoint, and B_Input is the absolute quantification of the input inoculum of the barcoded strain with mutated gene B.

AND/OR

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tubes	Eppendorf	22600028	Procure from any manufacturer
16 mL culture tubes	MidSci	8599	Procure from any manufacturer
5-200 μL pipette tips	RAININ	30389241	Procure alternative tip brands with caution based on manufacturing quality
5-50 μL multichannel pipette	RAININ	17013804	Use alternative multichannel pipettes with caution
Agarose	ThermoFisher Scientific	BP160-500	Procure from any manufacturer
BLAST Analysis	NCBI	N/A	https://blast.ncbi.nlm.nih.gov/Blast.cgi
C1000 Touch Thermocycler with 96-Deep Well Reaction Module	Bio Rad	1851197	Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.
Chemically competent DH5α	Invitrogen	18258012	Procure from any manufacturer or prepare yourself
Chloramphenicol	ThermoFisher Scientific	BP904-100	Procure from any manufacturer
Cytation5 Microplate reader	BioTek	CYT5MF	Procure from any manufacturer, use any system capable of accurately quantifying DNA
Data Analysis Software (QuantaSoft and QuantaSoft Data Analysis Pro)	Bio Rad	N/A	Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.
ddPCR 96-Well Plates	Bio Rad	12001925	Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.
ddPCR Droplet Reader Oil	Bio Rad	1863004	Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.
ddPCR Supermix for Probes (No dUTP)	Bio Rad	1863024	Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.
DG8 Cartridges for QX200/QX100 Droplet Generator	Bio Rad	1864008	Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.
DG8 Gaskets for QX200/QX100 Droplet Generator	Bio Rad	1863009	Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.
Droplet Generation Oil for Probes	Bio Rad	1863005	Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.
Kanamycin	ThermoFisher Scientific	BP906-5	Procure from any manufacturer
Luria-Bertani agar	ThermoFisher Scientific	BP1425-2	Procure from any manufacturer or make it yourself from agar, tryptone, yeast digest, and NaCl
Luria-Bertani broth	ThermoFisher Scientific	BP1426-2	Procure from any manufacturer or make it yourself from tryptone, yeast digest, and NaCl
PCR Plate Heat Seal, foil, pierceable	Bio Rad	1814040	Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.
PCR Tubes	Eppendorf	951010022	Procure from any manufacturer
Petri dishes	ThermoFisher Scientific	FB0875712	Procure from any manufacturer
pPCR Script Cam SK+	Stratagene/Agilent	211192	No longer available commercially
Primer/Probe Design	IDT	N/A	https://www.idtdna.com/Primerquest/Home/Index
pSKAP and pSKAP_Barcodes	Addgene	Plasmid numbers 122702-122726	www.addgene.org
PX1 PCR Plate Sealer	Bio Rad	1814000	Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.
QX200 Droplet Generator	Bio Rad	1864002	Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.
QX200 Droplet Reader	Bio Rad	1864003	Must procure ddPCR supplies from Bio Rad. Alternatives are not yet available.
Salmonella typhimurium strain ATCC 14028s	ATCC	ATCC 14028s	www.atcc.org
Take3 Micro-Volume Plate	BioTek	TAKE3	Procure from any manufacturer, use any system capable of accurately quantifying DNA
Thermo Scientific FastDigest BamHI	ThermoFisher Scientific	FERFD0054	Procure from any manufacturer
Thermo Scientific FastDigest DpnI	ThermoFisher Scientific	FERFD1704	Procure from any manufacturer
Thermo Scientific FastDigest HindIII	ThermoFisher Scientific	FERFD0504	Procure from any manufacturer
Thermo Scientific GeneJet Gel Extraction and DNA Cleanup Micro Kit	ThermoFisher Scientific	FERK0832	Procure from any manufacturer
Thermo Scientific GeneJet Miniprep Kit	ThermoFisher Scientific	FERK0503	Procure from any manufacturer
Thermo Scientific Phusion High-Fidelity DNA Polymerase	ThermoFisher Scientific	F534L	Procure from any manufacturer
Thermo Scientific T4 DNA Ligase	ThermoFisher Scientific	FEREL0011	Procure from any manufacturer
Thermocycler	Bio Rad	1861096	Procure from any manufacturer
UVP Visi-Blue Transilluminator	ThermoFisher Scientific	UV95043301	Or other transiluminator that allows visualization of DNA
Water, Molecular Biology Grade	ThermoFisher Scientific	BP28191	Procure from any manufacturer