This research was supported by a Discovery Grant from the Natural Sciences &#38; Engineering Council of Canada (NSERC).

1. Generation of DNA Constructs
<ol>
	<li>Designing Oligomers for Homologous Recombination of the Target Gene
	<ol>
		<li>Design a single 55-72 bp forward oligomer as follows: (a) pick a 19-32 bp long nucleotide sequence that is homologous to a DNA sequence in the region 10-100 bp upstream of the 5&#39; start site of the chloramphenicol acetyltransferase gene in the commercial pBAD33 plasmid,32 and (b) select a 36-54 bp long nucleotide sequence homologous to a region 10-150 bp downstream of ...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+diversity+of+prokaryotic+type+IV+secretion+systems.">Biological diversity of prokaryotic type IV secretion systems.</a> Microbiol Mol Biol Rev. 73 (4), 775-808 (2009).</li><li>Lawley, T. D., Klimke, W. A., Gubbins, M. J., Frost, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F+factor+conjugation+is+a+true+type+IV+secretion+system.">F factor conjugation is a true type IV secretion system.</a> FEMS Microbiol Lett. 224 (1), 1-15 (2003).</li><li>Lederberg, J., Tatum, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Recombination+in+Escherichia+Coli.">Gene Recombination in Escherichia Coli.</a> Nature. 158 (4016), 558-558 (1946).</li><li>Smillie, C., Garcillan-Barcia, M. P., Francia, M. V., Rocha, E. P. C., 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> Microbiol Mol Biol Rev. 74 (3), 434-452 (2010).</li><li>Willetts, N., Skurray, 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+Conjugation+System+of+F-Like+Plasmids.">The Conjugation System of F-Like Plasmids.</a> Annu Rev Genet. 14 (1), 41-76 (1980).</li><li>Frost, L. S., Ippen-Ihler, K., Skurray, R. 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J., Atmakuri, K., Krishnamoorthy, V., Jakubowski, S., Cascales, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+Architecture,+and+Function+of+Bacterial+Type+Iv+Secretion+Systems.">Biogenesis, Architecture, and Function of Bacterial Type Iv Secretion Systems.</a> Annu Rev Microbiol. 59 (1), 451-485 (2005).</li><li>Christie, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+IV+secretion:+the+Agrobacterium+VirB/D4+and+related+conjugation+systems.">Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems.</a> Biochim Biophys Acta - Mol Cell Res. 1694 (1-3), 219-234 (2004).</li><li>Bhatty, M., Laverde Gomez, J. a., Christie, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+bacterial+type+IV+secretion+lexicon.">The expanding bacterial type IV secretion lexicon.</a> Res Microbiol. 164 (6), 620-639 (2013).</li><li>Christie, P. J., Cascales, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+dynamic+properties+of+bacterial+Type+IV+secretion+systems+(Review).">Structural and dynamic properties of bacterial Type IV secretion systems (Review).</a> Mol Membr Biol. 22 (1-2), 51-61 (2005).</li><li>Christie, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+IV+secretion:+Intercellular+transfer+of+macromolecules+by+systems+ancestrally+related+to+conjugation+machines.">Type IV secretion: Intercellular transfer of macromolecules by systems ancestrally related to conjugation machines.</a> Mol Microbiol. 40 (2), 294-305 (2001).</li><li>Christie, P. J., Whitaker, N., Gonz&#225;lez-Rivera, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+and+structure+of+the+bacterial+type+IV+secretion+systems.">Mechanism and structure of the bacterial type IV secretion systems.</a> Biochim Biophys Acta. 1843 (8), 1578-1591 (2014).</li><li>Cascales, 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+type+VI+secretion+toolkit.">The type VI secretion toolkit.</a> EMBO Rep. 9, 735 (2008).</li><li>Silverman, J. M., Brunet, Y. R., Cascales, E., Mougous, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Regulation+of+the+Type+VI+Secretion+System.">Structure and Regulation of the Type VI Secretion System.</a> Annu Rev Microbiol. 66 (1), 453-472 (2012).</li><li>Chandran, V., 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=Structure+of+the+outer+membrane+complex+of+a+type+IV+secretion+system.">Structure of the outer membrane complex of a type IV secretion system.</a> Nature. 462 (7276), 1011-1015 (2009).</li><li>Rivera-Calzada, 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=Structure+of+a+bacterial+type+IV+secretion+core+complex+at+subnanometre+resolution.">Structure of a bacterial type IV secretion core complex at subnanometre resolution.</a> EMBO J. 32 (8), 1195-1204 (2013).</li><li>Waksman, G., Fronzes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+architecture+of+bacterial+type+IV+secretion+systems.">Molecular architecture of bacterial type IV secretion systems.</a> Trends Biochem Sci. 35, 691 (2010).</li><li>Fronzes, R., Christie, P. J., Waksman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+biology+of+type+IV+secretion+systems.">The structural biology of type IV secretion systems.</a> Nat Rev Microbiol. 7 (10), 703-714 (2009).</li><li>Kaplan, 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=Probing+a+cell-embedded+megadalton+protein+complex+by+DNP-supported+solid-state+NMR.">Probing a cell-embedded megadalton protein complex by DNP-supported solid-state NMR.</a> Nat Methods. 12 (7), 5-9 (2015).</li><li>Guyer, M. S., Reed, R. R., Steitz, J. A., Low, K. B. <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+a+sex-factor-affinity+site+in+E.+coli+as+gamma+delta.">Identification of a sex-factor-affinity site in E. coli as gamma delta.</a> Cold Spring Harb Symp Quant Biol. 45, 135-140 (1981).</li><li>Anthony, K. G., Sherburne, C., Sherburne, R., Frost, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+pilus+in+recipient+cell+recognition+during+bacterial+conjugation+mediated+by+F-like+plasmids.">The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F-like plasmids.</a> Mol Microbiol. 13 (6), 939-953 (1994).</li><li>Jobling, M. G., Holmes, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+vectors+with+the+pl5a+replicon,+kanamycin+resistance,+inducible+lacZ&#945;+and+pUC18+or+pUC19+multiple+cloning+sites.">Construction of vectors with the pl5a replicon, kanamycin resistance, inducible lacZ&#945; and pUC18 or pUC19 multiple cloning sites.</a> Nucleic Acids Res. 18 (17), 5315 (1990).</li><li>Guzman, L. M., Belin, D., Carson, M. J., Beckwith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+regulation,+modulation,+and+high-level+expression+by+vectors+containing+the+arabinose+P(BAD)+promoter.">Tight regulation, modulation, and high-level expression by vectors containing the arabinose P(BAD) promoter.</a> J Bacteriol. 177 (14), 4121-4130 (1995).</li><li>Yu, 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=An+efficient+recombination+system+for+chromosome+engineering+in+Escherichia+coli.">An efficient recombination system for chromosome engineering in Escherichia coli.</a> Proc Natl Acad Sci U S A. 97 (11), 5978-5983 (2009).</li><li>Lawley, T. D., Gilmour, M. W., Gunton, J. E., Standeven, L. J., 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=Functional+and+Mutational+Analysis+of+Conjugative+Transfer+Region+1+(Tra1)+from+the+IncHI1+Plasmid+R27.">Functional and Mutational Analysis of Conjugative Transfer Region 1 (Tra1) from the IncHI1 Plasmid R27.</a> J Bacteriol. 184 (8), 2173-2180 (2002).</li><li>Elton, T. C., Holland, S. J., Frost, L. S., Hazes, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F-Like+Type+IV+Secretion+Systems+Encode+Proteins+with+Thioredoxin+Folds+That+Are+Putative+DsbC+Homologues.">F-Like Type IV Secretion Systems Encode Proteins with Thioredoxin Folds That Are Putative DsbC Homologues.</a> J Bacteriol. 187 (24), 8267-8277 (2005).</li><li>Hazes, B., Frost, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+systems+biology+approach+to+study+type+II/IV+secretion+systems.">Towards a systems biology approach to study type II/IV secretion systems.</a> Biochim Biophys Acta. 1778, 1839-1850 (2008).</li><li>Lento, C., Ferraro, M., Wilson, D., Audette, G. F., Tsolis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDX-MS+and+deletion+analysis+of+the+type+4+secretion+system+protein+TraF+from+the+Escherichia+coli+F+plasmid.">HDX-MS and deletion analysis of the type 4 secretion system protein TraF from the Escherichia coli F plasmid.</a> FEBS Lett. 590 (3), 376-386 (2016).</li><li>Audette, G. F., Van Schaik, E. J., Hazes, B., Irvin, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-binding+protein+nanotubes:+Learning+from+nature's+nanotech+examples.">DNA-binding protein nanotubes: Learning from nature's nanotech examples.</a> Nano Lett. 4, 1897-1902 (2004).</li><li>Harris, R. L., Silverman, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tra+proteins+characteristic+of+F-like+type+IV+secretion+systems+constitute+an+interaction+group+by+yeast+two-hybrid+analysis.">Tra proteins characteristic of F-like type IV secretion systems constitute an interaction group by yeast two-hybrid analysis.</a> J Bacteriol. 186 (16), 5480-5485 (2004).</li><li>Moore, 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=Characterization+of+the+F-Plasmid+Conjugative+Transfer+Gene+traU.">Characterization of the F-Plasmid Conjugative Transfer Gene traU.</a> J Bacteriol. 172 (8), 4263-4270 (1990).</li><li>Anthony, K. G., Sherburne, C., Sherburne, R., Frost, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+pilus+in+recipient+cell+recognition+during+bacterial+conjugation+mediated+by+F-like+plasmids.">The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F-like plasmids.</a> Mol Microbiol. 13 (6), 939-953 (1994).</li><li>Klimke, W. A., Frost, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+the+role+of+the+transfer+gene,+traN,+of+the+F+and+R100-1+plasmids+in+mating+pair+stabilization+during+conjugation.">Genetic analysis of the role of the transfer gene, traN, of the F and R100-1 plasmids in mating pair stabilization during conjugation.</a> J Bacteriol. 180 (16), 4036-4043 (1998).</li><li>Jiang, W., Marraffini, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas:+New+Tools+for+Genetic+Manipulations+from+Bacterial+Immunity+Systems.">CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems.</a> Annu Rev Microbiol. 69 (1), 209-228 (2015).</li><li>Dahlberg, C., Bergstrom, M., Andreasen, M., Christensen, B. B., Molin, S., Hermansson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecies+bacterial+conjugation+by+plasmids+from+marine+environments+visualized+by+gfp+expression.">Interspecies bacterial conjugation by plasmids from marine environments visualized by gfp expression.</a> Mol Biol Evol. 15 (4), 385-390 (1998).</li></ol>

The authors declare that they have no competing financial interests.

Bacterial conjugation process provides a means by which bacteria can spread genes providing an evolutionary advantage for growth in challenging environments, such as the spread of antibiotic resistance markers. Because many of the conjugative plasmids are so large,12 functional studies on the proteins involved in assembly of the transfer apparatus through mutation of target genes on the conjugative plasmid itself are unwieldy. The protocols detailed herein provide a means by which one can more readily assess t...

The transfer of genes and proteins at the micro-organismal level plays a central role in bacterial survival and evolution as well as infection processes. The exchange of DNA between bacteria or between a bacterium and a cell can be achieved through transformation, conjugation or vector transduction.1,2 Conjugation is unique in comparison to transformation and transduction in that during conjugation between gram-negative bacteria such as Escherichia coli, the transfer of DNA occurs in a donor-controlled fashion whereby a complex macromolecular system connects donor and recipient cells. Conjugation is also the most direct way in which bacterial cells interact with host cells to inject genes, proteins or chemicals in to host systems.3 Quite often, the transfer of such agents has remarkable effects on the host, ranging from pathogenesis and carcinogenesis to host evolution and adaptation. It has been shown that conjugative recombination increases the rate of adaptation 3-fold in bacteria with high mutation rates under conditions of environmental stress.4 Moreover, conjugation is by far the most common route through which antibiotic resistance genes in bacterial strains are spread.5,6
Microorganisms have evolved specialized secretion systems to support the transfer of macromolecules across cellular membranes; there are currently 9 types of secretion systems (TSSs) in gram negative bacteria that have been described: T1SS, T2SS, T3SS, T4SS, T5SS, T6SS, T7SS, as well as the Sec (secretion) and Tat (two-arginine translocation) pathways.7,8 Each type of secretion system is further divided into different subtypes, a necessity due to diversity of proteins and the distinctiveness of pathways involved, in different bacterial strains. For example, in the type IV secretion system (T4SS), the Ti and Cag systems facilitate effector transport whereas the F-plasmid, R27 and pKM101 T4SSs facilitate transfer of a conjugative plasmid.7,9,10 A detailed understanding of the mechanisms by which organisms assemble their respective secretion systems from their component proteins and share cellular contents with a recipient or their surrounding environment is an important factor in development of targeted strategies to combat pathogenic microorganisms and processes of cellular infection.
Following the initial identification bacterial conjugation in E. coli by Lederberg &#38; Tatum,11 a large number of mobile and conjugative plasmids have been identified and characterized.12 Such mobile plasmids show considerable range is size (from 1 to over 200 kilobases (kb)), however all mobile plasmids contain a relaxase, which recognizes the origin of transfer (oriT) thereby enabling transmission of the plasmid. Conjugative plasmids further encode genes for assembly of a functional T4SS as well as a type IV coupling protein.12 For example the 100 kb F plasmid of E. coli encodes all the conjugative genes within a 33.3 kB transfer (tra) region.13 The genes in the tra region of the F plasmid encode all proteins that facilitate pilus formation, mating pair formation (Mpf), DNA transfer and exclusion functions during conjugative plasmid transfer.10,14,15 A significant body of knowledge is available for conjugative T4SSs, however detailed structural studies of the conjugative proteins and complexes are only more recently becoming available.16-28
In order to assemble a comprehensive view of the conjugative process, a coupling of detailed structural studies to mutational analyses of conjugative transfer proteins is required. This can be achieved through conjugative mating assays. For the F plasmid, each protein encoded within the tra region plays a role in the F-mediated conjugation; therefore, the knockout/deletion of a transfer gene will abolish the conjugative capacity of the cell (Figure 1). While smaller mobile plasmids are more conducive to standard deletion procedures, for larger conjugative plasmids such as F, gene knockouts are more readily achieved via homologous recombination where the target gene is replaced with one conveying a distinct antibiotic resistance gene. In the current protocol, we employ homologous recombination to replace a transfer gene of interest with chloramphenicol acetyltransferase (CAT) in the 55 kb F plasmid derivative pOX38-Tc;29,30 the resultant knockout plasmid, pOX38-Tc &#916;gene::Cm, facilitates resistance to the presence of chloramphenicol (Cm) in the growth media. Donor cells harboring pOX38-Tc &#916;gene::Cm are unable to affect conjugative DNA transfer/mating as observed through the use of a mating assay; the mating efficiency of a pOX38-Tc &#916;gene::Cm donor cell and a normal recipient will decrease or, more often, be abolished. Conjugative transfer of the pOX38-Tc &#916;gene::Cm plasmid can be restored via a small recovery plasmid harboring the targeted transfer gene. This recovery plasmid can be one that provides constitutive expression, such as plasmid pK184 (pK184-gene),31 or one that provides inducible expression so long as that plasmid properly targets the gene to the correct location within the cell (cytoplasm or periplasm). Consequently, in mating assays between this new donor (harboring pOX38-Tc &#916;gene::Cm + pK184-gene plasmids) and a recipient cell, the mating efficiency is expected to restore to nearly that of a normal donor-recipient mating assay. This system enables one to probe the function of the knocked out gene through the generation of a series of pK184-gene constructs (deletions or point mutations) and testing each construct&#39;s ability to restore the mating capacity of the pOX38-Tc &#916;gene::Cm harboring donor cells.

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">GeneJet Plasmid Mini-Prep Kit</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">K0503</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">GeneJet Gel Extraction Kit</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">K0692</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">GeneJet PCR Purification Kit</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">K0702</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Q5 Site-Directed Mutagenesis Kit</td>
			<td>New England Biolabs</td>
			<td>E0554S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Broad Range DNA Ladder</td>
			<td>New England Biolabs</td>
			<td>N0303A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Petri Dishes</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">FB0875713</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Electroporator</td>
			<td style="width:165px;">Eppendorf</td>
			<td style="width:131px;">4309000027</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Electroporation cuvettes</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">FB101</td>
			<td style="width:212px;">Cuvettes have a 1 mm gap.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Enzymes</td>
			<td style="width:165px;"></td>
			<td style="width:131px;"></td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">AvaI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0152S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">EcoRI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0101S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">HindIII</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0104L</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">NdeI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0111S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phusion DNA Polymerase</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">M0530L</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">T4 DNA Ligase</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">M0202S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DpnI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0176S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antibiotics</td>
			<td style="width:165px;"></td>
			<td style="width:131px;"></td>
			<td style="width:212px;">Final Concentrations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Chloramphenicol (Cm)</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">BP904-100</td>
			<td style="width:212px;">20 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Kanamycin (Km)</td>
			<td style="width:165px;">BioBasic Inc.</td>
			<td style="width:131px;">DB0286</td>
			<td style="width:212px;">50 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Nalidixic acid (Nal)</td>
			<td style="width:165px;">Sigma-Aldrich</td>
			<td style="width:131px;">N8878-25G</td>
			<td style="width:212px;">10 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Rifampicin (Rif)</td>
			<td style="width:165px;">Calbiochem</td>
			<td style="width:131px;">557303</td>
			<td style="width:212px;">20 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tetracycline (Tc)</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">BP912-100</td>
			<td style="width:212px;">10 &#181;g/mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Streptomycin (Sm)</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">BP910-50</td>
			<td style="width:212px;">50 &#181;g/mL</td>
		</tr>
	</tbody>
</table>

conjugative mating assays for sequence-specific analysis of transfer proteins involved in bacterial conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

The process of F plasmid-driven bacterial conjugation is a coordinated process that involves transfer proteins within the tra region of the F-plasmid that assembles a T4SS to facilitate pilus synthesis and conjugative DNA transfer. The protein TraF (GenBank accession # BAA97961; UniProt ID P14497) is required for conjugative F-pilus formation.10,14,35-37 The protein contains a C-terminal thioredoxin-like domain, though it does not have the catalyt...

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Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

The overall goal of this mating assay is to assess the effects of mutations within the conjugative transfer gene on its ability to affect plasmid transfer within the context of a functional type four secretion complex. This method allows for asking protein specific questions, such as identifying regions of protein-protein interactions that occur between transfer proteins as they assemble a functional type four secretion system in bacterial conjugation. In this technique, genes on large conjugative plasmids are knocked out by homologous recombination.Gene function is assessed by providing the target gene to the knock-outs on a smaller plasmid. Generally, individuals new to this method will struggle because organization and set-up are key to successfully conduct this procedure. Proper experimental preparation is necessary to get interpretable results.Begin this protocol with preparation of the digested pBAD33 plasmid as described in the text protocol. Run each digest on an agarose gel. Using a UV cabinet and a sterile razor, cut the 2.8 kilo baseband that cotresponds to the cath sequence out of the gel.Work quickly to minimize the UV exposure of the DNA. Extract the DNA from the cut out gel slice using a gel extraction kit as per the manufacturer's protocol. To amplify the extracted cat cassette by polymerase chain reaction or PCR.Prepare the reaction mixture in a sterile PCR tube. Use primers that contain overhangs that are homologous to the target gene sequence for homologous recombination. Set-up a negative control that bears the same components, except replace the template DNA with double distilled water.Also, set-up a positive control using template DNA and primers that have been proven to work in a PCR reaction. Mix all the reaction contents gently by pipetting. Then, amplify the extracted cat cassette using the PCR parameters listed in the text protocol.Use only five microliters of each reaction to confirm the correct size of amplification via agarose gel electrophoresis. Purify the PCR amplicon using a PCR purification kit with the manufacturer's protocol. Add 300 nanograms of the amplified cat cassette into 50 microliters of electrocompetent DY330R cells, harboring the pOX38-Tc plasmid on ice.Gently pipette up and down to mix. Repeat this step using unmodified pBAD33 plasmid as a positive control. Then, transfer the cells to a pre-cooled one millimeter electroporation cuvette.Electroporate the cells at 1.8 kilovolts with a time constant of 5.5 milliseconds using an electroporator. Immediately, after applying the pulse, dilute the cells with one milliliter of SOC media and transfer to a fresh microfuge tube. Incubate the cells at 32 degrees Celsius for two hours.Next, aliquot 100 microliters of each sample unto agar plates containing 10 micrograms per milliliter tetracycline and 20 micrograms per milliliter chloramphenicol. Spread the aliquot over the plate using a sterile spreader. Incubate the plate upside down at 32 degrees celsius overnight.The next day, select for the successful recombinants. The cat cassette introduced into the cell will undergo homologous recombination with the gene of interest and create the pOX38-Tc chloramphenicol knock-out clone. Separate the electroporate 300 nanograms of pK184 gene or pK184 gene mutant plasmid into 50 microliters of electrocompotent DY330R-cells harboring the pOX38-Tc chloramphenicol knock-out plasmid as before.To generate the XK1200 donor cells perform conjugative mating followed by electroporation to generate XK1200 cells harporing both the chloramphenicol knock-out and the PK184 plasmid as described in the text protocol. Preparing overnight culture of these XK1200 donor cells in 20 milliliters of sterile LB with chloramphenicol and kanamycin. Also prepare MC4100 recipients cells in 50 milliliters of LB with 50 micrograms per milliliter of streptomycin using cells from a glycerol stock or from a single colony on ang agar plate.Grow the cultures at 37 degrees celsius, shaking at 200 rpm. Add glucose to a final concentration of 100 millimolar to all donor cells. The next day, make 1 in 70 dilutions from each overnight culture separately in two milliliters of sterile LB with the same antibiotics.Grow the cells to mid log phase at 37 degrees celsius with shaking at 200 rpm. Centrifuge the cell culture to pellet the cells and discard the supernatant. Then, wash the cell pellet once with cold sterile LB to remove the antibiotics.After centrifuging a second time as before we suspend the cells in two milliliters of cold sterile LB.In duplicate, aliquot 100 microliters of donor cells and 100 microliters of recipient cells to 800 microliters of sterile LB media for one milliliter total volume. Allow the cells to mate at 37 degrees celsius for one hour without shaking. After an hour, vortex the cells for 30 seconds to disrupt the mating pairs.Then, place the cells on ice for 10 minutes to prevent further mating. Using the mid log cultures and fresh sterile LB, prepare six serial dilutions of the donor and recipient cells from 1 and 100 to 1 and 10 million. On each of two halves of an agar plate containing naladixic acid, chloramphenicol, and kanamycin, spot 10 microliter aliquots of each dilution of XK1200 donor cells.Repeat spotting for the dilutions of the recipient MC4100 cells on agar plates containing streptomycin. Then, incubate the plates overnight at 37 degrees celsius. Next, using the vortex mixture and fresh sterile LB, prepare six dilutions of the transconjugants.Select for the transconjugant MC4100 cells that harbor the pOX38-Tc chloramphenicol knock-out bu spotting ten microliter aliquots of each dilution on each half of the agar plates containing streptomycin and chloramphenicol. Repeat for both duplicate mixtures. Then, incubate the plates overnight at 37 degrees celsius.Calculate the mating efficiency by counting the number of colonies from the same dilution spotting for each donor, recipient and transconjugants cells on their respective plates. Also, count the recipient colonies to test for any bias resulting from a larger number of transconjugants than recipients at that given dilution. Calculate the mating efficiency as the number of transconjugant colonies divided by the number of donor colonies, multiplied by 100 to obtain the the efficiency value per 100 donor cells.When the type four secretion system gene-tra-F is knocked out of the pOX38-Tc plasmid, it results in a loss of conjugative transfer of the pOX38-Tc traF chloramphenicol knock out plasmid from donor to recipient cells. However, when the traF gene is provided in trans by the PK184 traF plasmid an the donor cells, a recovery of conjugative transfer of the pOX38-Tc traF chloramphenicol knock-out plasmid is observed. When provided with dilution mutants of traF and the PK184 plasmid in the donor cells, loss of conjugative transfer of the pOX38-Tc traF chloramphenicol knock-out plasmid can be observed.Loss of conjugative transfer indicates the region of the protein important for protein-protein interactions within the conjugative type four secretion system. This region can then be probed in more detail by point mutations that affect the efficiency for transfer. Once mastered, this technique can be performed in a single work day with expected growth times.This protocol can be done on cells other than the ones demonstrated here. The critical aspect, at this point, is that, donor and recipient cells don't harbor the plasmid of interest. So that when you add the knock-out plasmid, you don't get conflicting results.While attempting this procedure it is important to be very organized as there are different growth conditions and antibiotics for donor, recipient, and transconjugate cells. Following this procedure, other methods such as crystallography, NMR, or mass spectrometry can be performed in order to obtain a detailed structural and functional picture of the protein of interest.

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13.6K ビュー.  York University. The overall goal of this mating assay is to assess the effects of mutations within the conjugative transfer gene on its ability to affect plasmid transfer within the context of a functional type four secretion complex. This method allows for asking protein specific questions, such as identifying regions of protein-protein interactions that occur between transfer proteins as they assemble a functional type four secretion system in bacterial conjugation. In this technique, genes on large conjug...