We thank Dr. K. Kamachi of the National Institute of Infectious Diseases (Japan) for providing pBP136 and Prof. Dr. H. Nojiri of the University of Tokyo (Japan) for providing pCAR1. We are also grateful to Professor Dr. Molin S&#248;len of the Technical University of Denmark for providing pJBA28. This work was supported by JSPS KAKENHI (Grant Numbers 15H05618 and 15KK0278) to MS (https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15H05618/, https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15KK0278/).

1. Preparation of a Donor with Green Fluorescent Protein (GFP)- and Kanamycin Resistance Gene-Tagged Plasmids
<ol>
	<li>Introduction of marker genes into the target plasmid pBP136 
	Note: The goal of this protocol is to generate pBP136::gfp. The bacterial strains and plasmids used in this study are listed in Table 1.
	<ol>
		<li>Grow cultures of Escherichia coli DH10B harboring pBP13617 in 5 mL of sterile Luria br...

<ol>
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P., Alvarado, A., de la Cruz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+bacterial+plasmids+based+on+mobility+and+plasmid+population+biology.">Identification of bacterial plasmids based on mobility and plasmid population biology.</a> FEMS Microbiology Reviews. 35 (5), 936-956 (2011).</li><li>Smillie, C., Garcill&#225;n-Barcia, M. P., Francia, M. V., Rocha, E. P., de la Cruz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobility+of+plasmids.">Mobility of plasmids.</a> Microbiology and Molecular Biology Reviews. 74 (3), 434-452 (2010).</li><li>Garcill&#225;n-Barcia, M. P., Francia, M. V., de la Cruz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diversity+of+conjugative+relaxases+and+its+application+in+plasmid+classification.">The diversity of conjugative relaxases and its application in plasmid classification.</a> FEMS Microbiology Reviews. 33 (3), 657-687 (2009).</li><li>Shintani, M., 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=Recipient+range+of+IncP-7+conjugative+plasmid+pCAR2+from+Pseudomonas+putida+HS01+is+broader+than+from+other+Pseudomonas+strains.">Recipient range of IncP-7 conjugative plasmid pCAR2 from Pseudomonas putida HS01 is broader than from other Pseudomonas strains.</a> Biotechnology Letters. 27 (23-24), 1847-1853 (2005).</li><li>Yanagida, 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=Comparisons+of+the+transferability+of+plasmids+pCAR1,+pB10,+R388,+and+NAH7+among+Pseudomonas+putida+at+different+cell+densities.">Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among Pseudomonas putida at different cell densities.</a> Bioscience, Biotechnology, and Biochemistry. 80 (5), 1020-1023 (2016).</li><li>Schuurmans, J. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ecological+factors+on+conjugal+transfer+of+chromium-resistant+plasmid+in+Escherichia+coli+isolated+from+tannery+effluent.">Effect of ecological factors on conjugal transfer of chromium-resistant plasmid in Escherichia coli isolated from tannery effluent.</a> Biotechnology and Applied Biochemistry. 102 (1-6), 5-20 (2002).</li><li>Sakuda, 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=Divalent+cations+increase+the+conjugation+efficiency+of+the+incompatibility+P-7+group+plasmid+pCAR1+among+different+Pseudomonas+hosts.">Divalent cations increase the conjugation efficiency of the incompatibility P-7 group plasmid pCAR1 among different Pseudomonas hosts.</a> Microbiology. 164 (1), 20-27 (2018).</li><li>Corliss, T. L., Cohen, P. S., Cabelli, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R-plasmid+transfer+to+and+from+Escherichia+coli+strains+Isolated+from+human+fecal+samples.">R-plasmid transfer to and from Escherichia coli strains Isolated from human fecal samples.</a> Applied and Environmental Microbiology. 41 (4), 959-966 (1981).</li><li>Zhong, X., Krol, J. E., Top, E. M., Krone, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accounting+for+mating+pair+formation+in+plasmid+population+dynamics.">Accounting for mating pair formation in plasmid population dynamics.</a> Journal of Theoretical Biology. 262 (4), 711-719 (2010).</li><li>Gilmour, M. W., Lawley, T. D., Taylor, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cytology+of+bacterial+conjugation.">The cytology of bacterial conjugation.</a> EcoSal Plus. 2004, (2004).</li><li>Minoia, M., 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=Stochasticity+and+bistability+in+horizontal+transfer+control+of+a+genomic+island+in+Pseudomonas.">Stochasticity and bistability in horizontal transfer control of a genomic island in Pseudomonas.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (52), 20792-20797 (2008).</li><li>Kamachi, 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=Plasmid+pBP136+from+Bordetella+pertussis+represents+an+ancestral+form+of+IncP-1beta+plasmids+without+accessory+mobile+elements.">Plasmid pBP136 from Bordetella pertussis represents an ancestral form of IncP-1beta plasmids without accessory mobile elements.</a> Microbiology. 152 (12), 3477-3484 (2006).</li><li>Simon, R., Priefer, U., P&#252;hler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+broad+host+range+mobilization+system+for+in+vivo+genetic+engineering:+transposon+mutagenesis+in+gram+negative+bacteria.">A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria.</a> Nature Biotechnology. 1 (9), 784-791 (1983).</li><li>Andersen, J. B., 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=New+unstable+variants+of+green+fluorescent+protein+for+studies+of+transient+gene+expression+in+bacteria.">New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria.</a> Applied and Environmental Microbiology. 64 (6), 2240-2246 (1998).</li><li>Shintani, M., 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=Characterization+of+the+replication,+maintenance,+and+transfer+features+of+the+IncP-7+plasmid+pCAR1,+which+carries+genes+involved+in+carbazole+and+dioxin+degradation.">Characterization of the replication, maintenance, and transfer features of the IncP-7 plasmid pCAR1, which carries genes involved in carbazole and dioxin degradation.</a> Applied and Environmental Microbiology. 72 (5), 3206-3216 (2006).</li><li>Shintani, M., 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=Single-cell+analyses+revealed+transfer+ranges+of+IncP-1,+IncP-7,+and+IncP-9+plasmids+in+a+soil+bacterial+community.">Single-cell analyses revealed transfer ranges of IncP-1, IncP-7, and IncP-9 plasmids in a soil bacterial community.</a> Applied and Environmental Microbiology. 80 (1), 138-145 (2014).</li><li>Jarvis, B., Wilrich, C., Wilrich, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconsideration+of+the+derivation+of+most+probable+numbers,+their+standard+deviations,+confidence+bounds+and+rarity+values.">Reconsideration of the derivation of most probable numbers, their standard deviations, confidence bounds and rarity values.</a> Journal of Applied Microbiology. 109 (5), 1660-1667 (2010).</li><li>Haagensen, J. A., Hansen, S. K., Johansen, T., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+horizontal+transfer+of+mobile+genetic+elements.">In situ detection of horizontal transfer of mobile genetic elements.</a> FEMS Microbiology Ecology. 42 (2), 261-268 (2002).</li><li>Herrero, M., de Lorenzo, V., Timmis, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon+vectors+containing+non-antibiotic+resistance+selection+markers+for+cloning+and+stable+chromosomal+insertion+of+foreign+genes+in+gram-negative+bacteria.">Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria.</a> Journal of Bacteriology. 172 (11), 6557-6567 (1990).</li><li>Bagdasarian, M., 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=Specific-purpose+plasmid+cloning+vectors.+II.+Broad+host+range,+high+copy+number,+RSF1010-derived+vectors,+and+a+host-vector+system+for+gene+cloning+in+Pseudomonas.">Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas.</a> Gene. 16 (1-3), 237-247 (1981).</li><li>Maeda, 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=Complete+nucleotide+sequence+of+carbazole/dioxin-degrading+plasmid+pCAR1+in+Pseudomonas+resinovorans+strain+CA10+indicates+its+mosaicity+and+the+presence+of+large+catabolic+transposon+Tn4676.">Complete nucleotide sequence of carbazole/dioxin-degrading plasmid pCAR1 in Pseudomonas resinovorans strain CA10 indicates its mosaicity and the presence of large catabolic transposon Tn4676.</a> Journal of Molecular Biology. 326 (1), 21-33 (2003).</li><li>Takahashi, Y., Shintani, M., Yamane, H., Nojiri, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complete+nucleotide+sequence+of+pCAR2:+pCAR2+and+pCAR1+were+structurally+identical+IncP-7+carbazole+degradative+plasmids.">The complete nucleotide sequence of pCAR2: pCAR2 and pCAR1 were structurally identical IncP-7 carbazole degradative plasmids.</a> Bioscience, Biotechnology, and Biochemistry. 73 (3), 744-746 (2009).</li></ol>

Here, we present a high-resolution protocol for detecting differences in conjugation frequency under different conditions, using a MPN method to estimate the number of transconjugants. One important step in the protocol is diluting the mixture of donor and recipient after mating until no transconjugants grow. Another step is adding high concentrations of antibiotics to the selective liquid medium to prevent further conjugation. These procedures can reduce the background caused by further conjugation in the selective medi...

Bacterial conjugation of mobile genetic elements, conjugative plasmids, and integrative and conjugative elements (ICEs) is important for the horizontal spread of genetic information. It can promote rapid bacterial evolution and adaptation and transmit multidrug resistance genes1,2. The conjugation frequency can be affected by proteins encoded on the conjugative elements for mobilization of DNA (MOB) and mating pair formation (MPF), including sex pili, which are classified according to MOB and MPF type3,4,5. It can also be affected by the donor and recipient pair6 and the growth conditions of the cells7,8,9,10,11,12 (growth rate, cell density, solid surface or liquid medium, temperature, nutrient availability, and the presence of cations). To understand how the conjugative elements spread among bacteria, it is necessary to compare conjugation frequency in detail.
The conjugation frequency between donor and recipient pairs after mating are usually estimated by conventional methods as follows. (i) First, the numbers of donor and recipient colonies are counted; (ii) then, the recipient colonies, which received the conjugative elements (= transconjugants) are counted; (iii) and finally, the conjugation frequency is calculated by dividing the colony forming units (CFU) of the transconjugants by those of the donor and/or recipient13. However, when using this method, the background is high due to additional conjugation events that can also occur on the selective plates used to obtain transconjugants when the cell density is high10. Therefore, it is difficult to detect small differences in frequency (below a 10-fold difference). We recently introduced a most probable number (MPN) method using liquid medium containing a higher concentration of antibiotics. This method reduced the background by inhibiting further conjugation in selective medium; thus, the conjugation frequency could be estimated with higher resolution.
Conjugation can be divided into three steps: (1) attachment of the donor-recipient pair (2) initiation of conjugative transfer, and (3) dissociation of the pair14. During steps (1) and (3), there is physical interaction between the donor and recipient cells; thus, cell density and the environmental conditions can influence these steps, although the features of the sex pili are also important. Step (2) is likely regulated by the expression of several genes involved in conjugation in response to external changes, which could be affected by various features of the plasmid, donor, and recipient. Although the physical attachment or detachment of donor-recipient pairs can be mathematically simulated using an estimation of cells as particles, the frequency of step (2) should be experimentally measured. There have been a few reports on direct observations of how often donors can initiate conjugation [step (2)] using fluorescence microscopy15,16; however, these methods are not high-throughput because a large number of cells must be monitored. Therefore, we developed a new method to estimate the probability of the occurrence of step (2) by using fluorescence activated cell sorting (FACS). Our method can be applied to any plasmid, without identification of the essential genes for conjugation.

<table>
	<tbody>
		<tr>
			<td>MoFlo XDP</td>
			<td>Beckman-Coulter</td>
			<td>ML99030</td>
			<td>FACS</td>
		</tr>
		<tr>
			<td>IsoFlow</td>
			<td>Beckman-Coulter</td>
			<td>8599600</td>
			<td>Sheath solution</td>
		</tr>
		<tr>
			<td>Fluorospheres (10 &#956;m)</td>
			<td>Beckman-Coulter</td>
			<td>6605359</td>
			<td>beads to set up the FACS</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Yamato Scientific Co. Ltd</td>
			<td>211197-IC802</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-VIS Spectrophotometer UV-1800</td>
			<td>SIMADZU Corporation</td>
			<td>UV-1800</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>NIPPON Genetics Co, Ltd</td>
			<td>TR5003</td>
			<td></td>
		</tr>
		<tr>
			<td>microplate type Petri dish</td>
			<td>AXEL</td>
			<td>1-9668-01</td>
			<td>for validation of sorting</td>
		</tr>
		<tr>
			<td>membrane filter</td>
			<td>ADVANTEC</td>
			<td>C045A025A</td>
			<td>for filter mating</td>
		</tr>
		<tr>
			<td>pippettes</td>
			<td>Nichiryo CO. Ltd</td>
			<td>00-NPX2-20, 
			00-NPX2-200, 
			00-NPX2-1000</td>
			<td>0.5-10 &#956;L, 20-200 &#956;L, 100-1000 &#956;L</td>
		</tr>
		<tr>
			<td>multi-channel pippetes</td>
			<td>Nichiryo CO. Ltd</td>
			<td>00-NPM-8VP, 
			00-NPM-8LP</td>
			<td>0.5-10 &#956;L, 20-200 &#956;L</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>BD Difco</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD Difco</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S-5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Nakarai tesque</td>
			<td>01162-15</td>
			<td></td>
		</tr>
		<tr>
			<td>rifampicin</td>
			<td>Wako</td>
			<td>185-01003</td>
			<td></td>
		</tr>
		<tr>
			<td>gentamicin</td>
			<td>Wako</td>
			<td>077-02974</td>
			<td></td>
		</tr>
		<tr>
			<td>kanamycin</td>
			<td>Wako</td>
			<td>115-00342</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>AXEL</td>
			<td>3-1491-51</td>
			<td>JPND90-15</td>
		</tr>
		<tr>
			<td>microtubes</td>
			<td>Fukaekasei</td>
			<td>131-815C</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL disposable spinner flask</td>
			<td>Corning</td>
			<td>CLS3578</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-resolution comparison of bacterial conjugation frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

Comparison of conjugation frequency by the MPN method
In our previous report, we compared the conjugation frequencies of pBP136::gfp and pCAR1::gfp in three-fold diluted LB (1/3 LB) liquid medium with different stirring rates after a 45 min mating using 125 mL spinner flasks10. We compared the conjugation frequencies of pBP136::gfp and pCAR1::gfp with 106 CFU/mL...

Watch this Scientific Journal Video about High-Resolution Comparison of Bacterial Conjugation Frequencies at JoVE.com

High-Resolution Comparison of Bacterial Conjugation Frequencies

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

This method can help answer key questions in the microbiology field about how often plasmid DNA can be spread among different bacteria. The main advantage of this technique is that by reducing the background, we can detect small differences in the conjugation frequency with a high resolution. Demonstrating the procedure will be Kosuke Yanagiya, a graduate student from my laboratory.To calculate the conjugation frequency by most probable number, filter mate one milliliter from an overnight donor culture with one milliliter from an overnight recipient culture for 45 minutes at 30 degrees Celsius, as just demonstrated. While the co-culture is incubating, serially dilute the original donor and recipient cultures for plating in triplicate onto donor Luria broth, or LB, plus kanamycin, or recipient LB plus gentamycin plates for a two-day culture at 30 degrees Celsius. At the end of the incubation, resuspend the culture on the filter in sterile LB containing kanamycin and gentamycin for serial dilution in a 96-well cell culture plate in quadruplicate.After two days at 30 degrees Celsius, manually count the colony-forming units, or CFUs, on the donor and recipient agar plates and the number of wells in which the transconjugants grow by light microscopy. To calculate the most probable number and its deviation, open the MPN calculation program. Enter the name and date of the experiment, the number of the test series, and the maximum number of dilutions in row seven of the program sheet of the spreadsheet file.In the automatically-produced input data tables, enter the formula in the dilution factor D column, 0.01 in the volume in milliliters or GW column, and four in the number of tubes N column. Enter the number of wells in which the transconjugants grew at each sample dilution and click calculate results to obtain the results as most probable number per milliliter, and the upper and lower 95%confidence limits. Then divide the number of transconjugants by the number of donor and recipient colony forming units per milliliter to calculate the conjugation frequency of the plasmids.In this representative experiment, the conjugation frequency of both plasmids increased at higher stirring rates with a maximum difference in conjugation frequency observed between zero and 400 RPM for cultures introduced to the pBP136:gfp plasmid, and between zero and 200 RPM for cultures that were introduced to the pCAR1:gfp plasmid. To determine the density of recipient cells required to compare the probability of conjugation, mating assays were performed with different donor and recipient densities. In this representative experiment, pBP136:gfp transconjugants were detected in 100%of the wells containing one times 10 to the third CFU of donor and one times 10 to the fifth to one times 10 to the seventh CFU of recipient, and those with one times 10 to the second CFU of donor and one times 10 to the sixth to 10 to the seventh CFU of recipient, indicating that the cell density was too high.Mating assays with 10 CFU of donor and 10 to the sixth or 10 to the fifth CFU of recipient, however, resulted in a decreased number of transconjugant positive wells. Thus, 10 to the fifth CFU of recipient was predicted to be required for mating with a single donor cell. Mating assays with pCAR1:gfp at different densities of donor and recipient strains demonstrated similar findings, although overall the percentages of transconjugant positive wells were much lower than those of pBP136:gfp.Assuming that the donor and recipient cells can attach to each other similarly, the probability of conjugation initiation for the pCAR1 donor was lower than that for the pBP136 donor. Based on these results, it was estimated that 10 to the seventh CFU of recipient was required for a single donor cell sorted by FACS. After counting the numbers of transconjugant positive wells, the percentage of transconjugant positive wells for pBP136:gfp was determined to be larger than that for pCAR1:gfp, demonstrating a more than 36-fold difference in the probability of donor initiated conjugation between these two plasmids.While attempting the procedure, it's important to remember to always dilute the bacterial mixtures carefully.

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10.5K Views.  Shizuoka University. This method can help answer key questions in the microbiology field about how often plasmid DNA can be spread among different bacteria. The main advantage of this technique is that by reducing the background, we can detect small differences in the conjugation frequency with a high resolution. Demonstrating the procedure will be Kosuke Yanagiya, a graduate student from my laboratory.To calculate the conjugation frequency by most probable number, filter mate one milliliter from an overni...