This work was supported by Public Health Service grants from the National Institute of Health, NIAID (grants A1065530 and Al124649), to K.A. Fields.

1. Design and Assembly of pSUmC-4.0 with Homology Arms Specific to the Gene of Interest
<ol>
	<li>Identify ~3 kb regions directly upstream and downstream of the gene targeted for deletion to serve as the 5&#39; and 3&#39; homology arms for homologous recombination (Figure 1).</li>
	<li>Design PCR primers to 1) amplify the 3 kb 5&#39; homology arm from chlamydial genomic DNA and 2) contain a 15&#8211;30 bp overhang specific to pSUmC-4.0 when digested at the Sall restriction enzyme site. Ens...

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Incidence+and+Prevalence+of+Selected+Curable+Sexually+Transmitted+Infections:+2008:+World+Health+Organization.">Global Incidence and Prevalence of Selected Curable Sexually Transmitted Infections: 2008: World Health Organization.</a> Sexual and Reproductive Health Matters. 20, 207-208 (2012).</li><li>Stephen, R. 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+Cellular+Paradigm+of+Chlamydial+Pathogenesis.">The Cellular Paradigm of Chlamydial Pathogenesis.</a> Trends in Microbiology. 11, 44-51 (2003).</li><li>Mueller, K. E., Plano, G. V., Fields, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Frontiers+in+Type+III+Secretion+Biology:+the+Chlamydia+Perspective.">New Frontiers in Type III Secretion Biology: the Chlamydia Perspective.</a> Infection and Immunity. 82, 2-9 (2014).</li><li>Seth-Smith, H. M. 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=Co-evolution+of+genomes+and+plasmids+within+Chlamydia+trachomatis+and+the+emergence+in+Sweden+of+a+new+variant+strain.">Co-evolution of genomes and plasmids within Chlamydia trachomatis and the emergence in Sweden of a new variant strain.</a> BMC Genomics. 10, 239 (2009).</li><li>Brothwell, J. A., Muramatsu, M. K., Zhong, G., Nelson, 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=Advances+and+obstacles+in+genetic+dissection+of+chlamydial+virulence.">Advances and obstacles in genetic dissection of chlamydial virulence.</a> Current Topics in Microbiology and Immunology. 412, 133-158 (2018).</li><li>McClure, E. E., 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=Engineering+of+obligate+Intracellular+bacteria:+progress,+challenges+and+paradigms.">Engineering of obligate Intracellular bacteria: progress, challenges and paradigms.</a> Nature Reviews Microbiology. 15, 544-558 (2017).</li><li>Rahnama, M., Fields, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Chlamydia:+current+approaches+and+impact+on+our+understanding+of+chlamydial+infection+biology.">Transformation of Chlamydia: current approaches and impact on our understanding of chlamydial infection biology.</a> Microbes and Infection. 20 (7-8), 445-450 (2018).</li><li>Kari, L., 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=Generation+of+targeted+Chlamydia+trachomatis+null+mutants.">Generation of targeted Chlamydia trachomatis null mutants.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 7189-7193 (2011).</li><li>LaBrie, S. 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=Transposon+Mutagenesis+in+Chlmaydia+trachomatis+Identifies+CT339+as+a+ComEC+Homolog+Important+for+DNA+Uptake+and+Later+Gene+Transfer.">Transposon Mutagenesis in Chlmaydia trachomatis Identifies CT339 as a ComEC Homolog Important for DNA Uptake and Later Gene Transfer.</a> mBio. 10 (4), 01343 (2019).</li><li>Johnson, C. M., Fisher, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific,+insertional+inactivation+of+incA+in+Chlamydia+trachomatis+using+a+group+II+intron.">Site-specific, insertional inactivation of incA in Chlamydia trachomatis using a group II intron.</a> PLoS One. 8, 83989 (2013).</li><li>Mueller, K. E., Wolf, K., Fields, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Deletion+by+Fluorescence-Reported+Allelic+Exchange+Mutagenesis+in+Chlamydia+trachomatis.">Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis.</a> mBio. 7, 01817 (2016).</li><li>Keb, G., Hayman, R., Fields, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floxed+cassette+Allelic+Exchange+Mutagenesis+Enables+Markerless+Gene+Deletion+in+Chlamydia+trachomatis+and+can+Reverse+Cassette-Induced+Polar+Effects.">Floxed cassette Allelic Exchange Mutagenesis Enables Markerless Gene Deletion in Chlamydia trachomatis and can Reverse Cassette-Induced Polar Effects.</a> Journal of Bacteriology. 200, 00479 (2018).</li><li>Yarmolinsky, M., Hoess, 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+legacy+of+Nat+Sternberg:+the+genesis+of+Cre-lox+technology.">THe legacy of Nat Sternberg: the genesis of Cre-lox technology.</a> Annual Review of Virology. 2, 25-40 (2015).</li><li>McKuen, M. J., Mueller, K. E., Bae, Y. S., Fields, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-Reported+Allelic+Exchange+Mutagenesis+Reveals+a+Role+for+Chlamydia+trachomatis+TmeA+in+Invasion+that+is+Independent+of+Host+AHNAK.">Fluorescence-Reported Allelic Exchange Mutagenesis Reveals a Role for Chlamydia trachomatis TmeA in Invasion that is Independent of Host AHNAK.</a> Infection and Immunity. 85 (12), 00640 (2017).</li><li>Song, L., 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=Chlamydia+trachomatis+Plasmid-Encoded+Pgp4+is+a+Transcriptional+Regulator+of+Virulence-Associated+Genes.">Chlamydia trachomatis Plasmid-Encoded Pgp4 is a Transcriptional Regulator of Virulence-Associated Genes.</a> Infection and Immunity. 81 (3), 636-644 (2013).</li><li>Silayeva, O., Barnes, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gibson+Assembly+facilitates+bacterial+allelic+exchange+mutagenesis.">Gibson Assembly facilitates bacterial allelic exchange mutagenesis.</a> Journal of Microbiological Methods. 144, 157-163 (2017).</li><li>Hackstadt, T., Scidmore, M., Rockey, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Metabolism+in+Chlamydia+trachomatis-Infected+Cells:+Directed+Trafficking+of+Golgi-Derived+Sphingolipids+to+the+Chlamydial+Inclusion.">Lipid Metabolism in Chlamydia trachomatis-Infected Cells: Directed Trafficking of Golgi-Derived Sphingolipids to the Chlamydial Inclusion.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (11), 4877-4881 (1995).</li><li>Jeffrey, B. M., Suchland, R. J., Eriksen, S. G., Sandoz, K. M., Rockey, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+phenotypic+characterization+of+in+vitro-generated+Chlamydia+trachomatis+recombinants.">Genomic and phenotypic characterization of in vitro-generated Chlamydia trachomatis recombinants.</a> BMC Microbiology. 13, 142 (2013).</li><li>Suchland, R. J., Bourillon, A., Denamur, E., Stamm, W. E., Rothstein, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rifampin-resistant+RNA+polymerase+mutants+of+Chlamydia+trachomatis+remain+susceptible+to+the+ansamycin+rifalazil.">Rifampin-resistant RNA polymerase mutants of Chlamydia trachomatis remain susceptible to the ansamycin rifalazil.</a> Antimicrobial Agents Chemotherapy. 49, 1120-1126 (2005).</li></ol>

The authors have no conflicts of interest to disclose.

The protocol described here for the generation of markerless gene deletions in C. trachomatis by FLAEM allows targeted deletion of nonessential genes and eliminates cassette-induced polar effects. The protocol relies upon careful design of 5&#39; and 3&#39; homology arms inserted into the pSUmC 4.0 suicide vector, efficient transformation of C. trachomatis, and careful screening of isolated mutant strains. Successful genome engineering via this method results in bacteria that are nonfluorescent and cont...

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease and represents a significant burden to human health. Over 100 million people are infected every year with C. trachomatis1. Approximately 70% of the infections in women are asymptomatic despite detrimental reproductive health effects, such as pelvic inflammatory disease, ectopic pregnancy, and/or infertility. Disease sequela are directly related to immunopathology initiated by C. trachomatis infection2. An efficacious vaccine has yet to be developed; therefore, understanding the function of bacterial virulence factors and other bacterial gene products is an important and urgent research question.
As intracellular bacteria, host cell invasion, intracellular replication, release of progeny, and evasion of host immunological responses are critical processes. C. trachomatis forms a parasitophorous membrane bound vacuole, termed an inclusion, for intracellular development. Establishment of the inclusion and many other critical processes are achieved by secretion of effector proteins via a type III secretion system (T3SS)3. Elucidating the functions of these secreted effectors was limited for many years due to the genetic intractability of C. trachomatis. Unlike E. coli, many classical cloning techniques are not applicable to Chlamydia. A few major limitations involve transformation efficiency, lack of counterselection reporters such as sacB, and plasmid maintenance. Whereas E. coli plasmids can generally be maintained indefinitely with an origin of replication and appropriate selective pressure, C. trachomatis plasmids requires an additional eight open reading frames (pgp1-8) for maintenance that are found on the native pL2 plasmid within the L2 serovar4.
In recent years there have been multiple genetic tools generated that accommodate Chlamydia&#39;s unique biology, yet there are still limitations5,6,7. Chemical mutagenesis by ethyl methanesulfonate (EMS) treatment can introduce missense mutations, or (less frequently) can result in nucleotide transitions introducing a premature stop codon to yield a nonsense mutation8. Transposon insertion is efficient for gene disruption, but current technology in Chlamydia research is laborious and time-consuming9. Both EMS treatment and transposon mutagenesis techniques generate random mutations and require rigorous screening methods to isolate mutant strains. A method to disrupt genes by insertion of group II introns (e.g., TargeTron) allows for directed mutagenesis; however, this method is limited by efficiency, and the insertion site is not always properly predicted10.
Fluorescence-reported allelic exchange mutagenesis (FRAEM) is a strategy used for targeted gene deletion coupled with insertion of a selection cassette providing antibiotic resistance and a fluorescence reporter11. Yet, FRAEM is complicated by the potential of cassette-induced polar effects on downstream genes, especially when targeting genes within polycistronic operons. Floxed cassette allelic exchange mutagenesis (FLAEM) is a novel genetic approach developed to alleviate the cassette-induced polar effects previously observed with the FRAEM selection cassette12. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette and restore expression of downstream genes. The selection cassette containing antibiotic resistance and green fluorescent protein (GFP) is reengineered with flanking loxP sites. These loxP sites can recombine in the presence of Cre recombinase and result in excision of the cassette from the genome13. This strategy has been shown to alleviate cassette induced polar effects when targeting tmeA for deletion12,14.
Both FRAEM and FLAEM methods utilize the same suicide vector, pSUmC 4.0, which can be conditionally maintained through inducible expression of pgp6. Expression of pgp6 has previously been shown to be necessary for plasmid retention and is therefore leveraged to control plasmid maintenance11,15. When C. trachomatis is grown in media supplemented with anhydrous tetracycline (aTc) to induce pgp6 expression, the vector is maintained. In the absence of aTc, the vector is lost. Targeted gene deletion is achieved through allelic exchange of the gene for the selection cassette. The 3 kb regions directly upstream and downstream of the targeted gene serve as homology arms for recombination. These arms are cloned into the pSUmC 4.0 vector flanking the selection cassette. Successful C. trachomatis transformation and recombination events are observed through fluorescence reporting. Expression of mCherry on the vector backbone and gfp within the selection cassette yield red and green fluorescent inclusions. Once aTc is removed from culture media, green-only inclusions indicate successful recombination events with the loss of the suicide vector and integration of the selection cassette into the bacterial genome.
FLAEM represents an extension of FRAEM via subsequent transformation of a Cre recombinase-expressing vector, pSU-Cre, into the newly created mutant strain. Cre recombinase facilitates recombination between loxP sites and excision of the selection cassette. Recombination events are indicated via fluorescence reporting. The pSU-Cre vector encodes mCherry; thus, successful transformation is indicated by addition of red fluorescence to gfp-expressing inclusions. Cultivation in the absence of selective pressure for the cassette results in Cre-mediated recombination at the loxP sites, and loss of the cassette is indicated by red-only inclusions. As with pSUmC-4.0, inducible expression of pgp6 is used to conditionally maintain pSU-Cre. Once aTc and antibiotic selection are removed, the plasmid is cured, and the resulting markerless deletion strain is nonfluorescent. This method addresses the issue of cassette-induced polar effects.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>KSE Scientific</td>
			<td>BMK-A1705</td>
			<td>Molecular Biology Grade</td>
		</tr>
		<tr>
			<td>Anhydrotetracycline hydrochloride</td>
			<td>ACROS Organics</td>
			<td>233131000</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 Buffer</td>
			<td></td>
			<td></td>
			<td>10 mM Tris pH 7.4, 50 mM Calcium Chloride Dihydrate</td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma</td>
			<td>C7902-500G</td>
			<td>Suitable for cell culture</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>7698-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>dam-/dcm- Competent E. coli</td>
			<td>New England BioLabs</td>
			<td>C2925H</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>ATCC</td>
			<td>4-X</td>
			<td>Sterile filtered cell culture tested</td>
		</tr>
		<tr>
			<td>Glutamic acid</td>
			<td>Sigma</td>
			<td>G8415-100G</td>
			<td>L-Glutamic acid</td>
		</tr>
		<tr>
			<td>Growth Media #1</td>
			<td></td>
			<td></td>
			<td>RPMI 1640 media supplemented with 10 % (vol/vol) heat-inactivated fetal bovine serum (FBS).</td>
		</tr>
		<tr>
			<td>Growth Media #2</td>
			<td></td>
			<td></td>
			<td>RPMI 1640 media supplemented with 10 % (vol/vol) heat-inactivated fetal bovine serum (FBS) and 1 &#181;g/mL cycloheximide</td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS) (1x)</td>
			<td>Gibco</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated Fetal Bovine Serum Qualified One Shot (FBS)</td>
			<td>Gibco</td>
			<td>A38402-02</td>
			<td></td>
		</tr>
		<tr>
			<td>McCoy Cells</td>
			<td>ATCC</td>
			<td>CRL-1696</td>
			<td></td>
		</tr>
		<tr>
			<td>Monarch Plasmid Miniprep Kit</td>
			<td>New England BioLabs</td>
			<td>T1010S</td>
			<td>Small scale DNA purification</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S3139-250G</td>
			<td>Sodium phosphate monobasic</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma</td>
			<td>S5136-500G</td>
			<td>Sodium phosphate dibasic</td>
		</tr>
		<tr>
			<td>NEB 10-beta Electrocompetent E. coli Cells</td>
			<td>New England BioLabs</td>
			<td>C3020K</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuilder HiFi DNA assembly Cloning Kit</td>
			<td>New England BioLabs</td>
			<td>E5520S</td>
			<td>Gibson Assembly Kit</td>
		</tr>
		<tr>
			<td>Penicillin G sodium salt</td>
			<td>Sigma</td>
			<td>P3032-10MU</td>
			<td>Bioreagent suitable for cell culture</td>
		</tr>
		<tr>
			<td>QIAGEN Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12162</td>
			<td>Large scale DNA purification</td>
		</tr>
		<tr>
			<td>Q5 Hot Start High-Fidelity DNA Polymerase</td>
			<td>New England BioLabs</td>
			<td>M0515</td>
			<td>Fragment PCR Polymerase</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium (1x)</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td>Containing 2mM L-glutamine</td>
		</tr>
		<tr>
			<td>Sall-HF</td>
			<td>New England BioLabs</td>
			<td>R3138S</td>
			<td></td>
		</tr>
		<tr>
			<td>Sbfl-HF</td>
			<td>New England BioLabs</td>
			<td>R3642S</td>
			<td></td>
		</tr>
		<tr>
			<td>Selection Media #1</td>
			<td></td>
			<td></td>
			<td>RMPI 10 % FBS, 1 &#181;g/mL cycloheximide, 500 &#181;g/mL spectinomycin, and 50 ng/mL anhydrous tetracycline dissolved in DMSO</td>
		</tr>
		<tr>
			<td>Selection Media #2</td>
			<td></td>
			<td></td>
			<td>RMPI 10 % FBS, 1 &#181;g/mL cycloheximide, 500 &#181;g/mL spectinomycin</td>
		</tr>
		<tr>
			<td>Selection Media #3</td>
			<td></td>
			<td></td>
			<td>RPMI 10 % FBS, 1 &#181;g/mL cycloheximide, 50 ng/mL aTc, and 0.6 &#181;g/mL penicillin</td>
		</tr>
		<tr>
			<td>Sodium Acetate Buffer Solution</td>
			<td>Sigma</td>
			<td>S7899-100ML</td>
			<td>3M</td>
		</tr>
		<tr>
			<td>SOC Outgrowth Medium</td>
			<td>New England BioLabs</td>
			<td>B9020SVIAL</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin dihydrochloride pentahydrate, Cell Culture Grade</td>
			<td>Alfa Aesar</td>
			<td>J61820</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S1888-1KG</td>
			<td>Bioreagent suitable for cell culture</td>
		</tr>
		<tr>
			<td>Sucrose-Phosphate-Glutamate Buffer (SPG)</td>
			<td></td>
			<td></td>
			<td>37.5g sucrose, 1.25 g Na2HPO4, 0.18 g NaH2PO4, 0.36 glutamic acid for 500 ml tissue culture grade water</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>AMRESCO</td>
			<td>0497-5KG</td>
			<td>Ultrapure grade</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (1x)</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>0.25%</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W3500-500ML</td>
			<td>Sterile-filtered, BioReagent, Suitable for cell culture</td>
		</tr>
	</tbody>
</table>

markerless gene deletion by floxed cassette allelic exchange mutagenesis in chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.

The method for markerless gene deletion in C. trachomatis using FLAEM is reliant upon careful cloning and transformation techniques. Successful allelic recombination is an essential first step and requires the identification and insertion of homology arms into the pSUmC-4.0 cloning vector (Figure 1). An essential second step for markerless gene deletion is removal of the fluorescence reporter and antibiotic selection cassette by Cre-lox genome editing, represented in <strong class="...

Watch this Scientific Journal Video about Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis at JoVE.com

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in ...

Floxed cassette allelic exchange mutagenesis is an important method because gene deletion is a valuable tool that can be utilized to understand the function of gene products. The main advantage of this technique is that it allows targeted gene deletion without inflicting polar effects on downstream genes. Identify approximately three kilobase regions directly upstream and downstream of the gene targeted for deletion to serve as the five prime and three prime homology arms for homologous recombination and assign primers as indicated in the text protocol.Amplify the three kilobase five prime and three prime homology arms from freshly extracted chlamydial genomic DNA by PCR in one or two reactions to yield enough fragment for later DNA assembly. After purifying and concentrating the PCR fragments as described in the text protocol, digest 500 nanograms of pSUmC-4.0 vector in a 20 microliter reaction with one unit of cell one and one unit of SBF one according to the manufacturer's protocol. Combine the digested and purified pSUmC-4.0 vector and both the five prime and three prime homology arm PCR products together at a ratio of 0.01 picomoles vector to 0.09 picomoles of each homology arm.Assemble the fragments in a DNA assembly reaction according to the manufacturer's protocol. Transform 50 microliters of electrocompetent E.coli with one microliter of the DNA assembly reaction. Plate the transformed E.coli onto LB agar plates containing 100 micrograms per milliliter spectinomycin.Incubate the plates at 37 degrees Celsius overnight. The next day, screen colonies for green and red fluorescence using an epifluorescence inverted microscope. A total of five to 30 colonies per agar plate is expected.Following an overnight liquid culture incubation of the selected colonies, confirm the insertion of each homology arm into the vector using the PCR screening primers to amplify each insertion. Seed McCoy cells which are mouse fibroblasts typically used for cultivating Chlamydia into two six-well plates as described in the text protocol. In a 1.5 milliliter tube, add C.trachomatis wild-type serovar L2 elementary bodies, or EBs, at a volume that is sufficient to infect 12 wells of a six-well plate at an MOI of two.Pellet the EBs at greater than 20, 000 times g for 30 minutes and discard the supernatant. Initiate chlamydial transformation by gently resuspending the EBs in 600 microliters of calcium chloride buffer. Then, add 12 micrograms of pSUmC-4.0 plus homology arms to the tube and gently mix the solution by flicking.Incubate the tube at room temperature for 30 minutes flicking to mix every 10 minutes. Add the transformation solution to 24 milliliters of HBSS and mix by gently pipetting. Transfer two milliliters of the inoculum to each well of confluent McCoy cells in the six-well plates.Infect by centrifugation at 900 times g for one hour at 20 degrees Celsius. Remove the inoculum and replace with two milliliters per well RPMI growth media number two. Incubate the plates at 37 degrees Celsius with 5%carbon dioxide.After a minimum of seven hours but no more than 12 hours, replace the media with two milliliters per well of selection media number one. Continue the incubation at 37 degrees Celsius for 48 hours from the time of infection. Harvest and passage the bacteria onto a fresh monolayer of McCoy cells by using a cell scraper to gently scrape the McCoy monolayer to lift the cells into the media.Transfer the material from each well into a two milliliter tube. Pellet the cell material at greater than 20, 000 times g in a microcentrifuge for 30 minutes at four degrees Celsius. After discarding the supernatant, resuspend the cell pellet in one milliliter of HBSS by gently pipetting.Pellet the cell debris at 200 times g for five minutes at four degrees Celsius. Transfer the supernatant into a fresh well of confluent McCoy cells. Add an additional one milliliter of HBSS to each well for a total volume of two milliliters per well.Infect by centrifugation at 900 times g for one hour at 20 degrees Celsius. Replace the inoculum with selection media number one immediately following infection. Continue passaging the bacteria onto a fresh monolayer of McCoy cells every 48 hours until red and green inclusions are detected using an epifluorescence inverted microscope 24 hours post-infection.Once red and green inclusions are detected, continue passaging the monolayer as before using selection media number two. Continue passaging the monolayer until green-only inclusions are detected 24 hours post-infection which indicates the loss of the pSUmC-4.0 vector and incorporation of the loxP selection cassette into the genome. Harvest and freeze the green-only inclusions in SPG.Enrich green-only inclusions to an MOI of between 0.5 and 1.0 and harvest the monolayers into SPG as described in the text. Obtain aliquots of 50 microliters in 1.5 milliliter tubes and freeze at minus 80 degrees Celsius. Seed a 384-well tissue culture plate with McCoy cells as described in the text protocol.Dilute an aliquot of green-only bacteria in HBSS to achieve approximately 50 EBs per 20 milliliters. Transfer the diluted inoculum to a reservoir tray. Remove the media from the 384-well plate of confluent McCoy cells by firmly flicking the entire plate upside down into a waste container.Using a multichannel pipette, add 50 microliters of C.trachomatis inoculum to each well. Infect by centrifugation at 900 times g for one hour at 20 degrees Celsius. Remove the inoculum by flicking and replace with 50 microliters per well of growth media number two.Incubate the plate at 37 degrees Celsius with 5%carbon dioxide. After incubating the plate for five to 12 days, identify individual wells with green fluorescent inclusions using an epifluorescence inverted microscope or a high-content screening platform. Harvest wells with green-only inclusions by scraping the monolayer with a P10 tip.Transfer the entire contents of the well into a tube containing two milliliters of HBSS. Gently mix and apply the two milliliters of inoculum to a fresh confluent McCoy monolayer in a six-well plate for infection. Enrich, freeze, and titer clonal populations.Confirm the deletion of targeted genes from clonally isolated C.trachomatis using quantitative PCR. Repeat the transformation process using the clonally isolated C.trachomatis FRAEM mutant, pSU-Cre vector, and selection media number three. Passage the monolayer until red-only inclusions are detected which indicates loss of the selection cassette.Clonally isolate red-only inclusions by limiting dilution in a 384-well plate as before. Enrich clonal populations by passaging until an MOI of 0.1. Once enriched, replace the selection media with growth media number two to initiate the loss of the pSU-Cre vector.Clonally isolate non-fluorescent bacteria as before. However, manually scan the plate with a Brightfield microscope to detect inclusions. Enrich and freeze the bacteria.Screen for the mutant strain using quantitative real-time PCR, Western blotting, and whole genome DNA sequencing as previously described. Representative data is shown in which tmeA is targeted for gene deletion. The C.trachomatis tmeaA deletion strain is generated using FRAEM and the C.trachomatis tmeA-lx strain is generated using FLAEM.Relative DNA copy numbers of tmeA and downstream tmeB, gfp contained on the pSUmC-4.0 selection cassette, and cre contained on the pSU-Cre vector are all shown. Both mutant strains contain a deletion of the tmeA locus. However, tmeA-lx does not contain the selection cassette as indicated by the absence of gfp DNA.The tmeA mutant strain has decreased expression of tmeB as shown by mRNA and protein levels. When FLAEM is used to generate the tmeA-lx mutant strain, that expression of tmeB is restored as detected by mRNA and protein levels. Careful construction of the pSUmC vector with homology arms is essential for targeted deletion of genes.Time should be taken to ensure accurate vector assembly prior to chlamydial transformation. FLAEM has provided researchers a tool to study specific genes and make targeted deletions without having decreased expression of off target genes which may confound studies. C.trachomatis deletion mutants generated by FLAEM can be utilized to study a variety of aspects of chlamydial biology including developmental and pathogenic mechanisms.

markerless-gene-deletion-floxed-cassette-allelic-exchange-mutagenesis

Research

JoVE Journal

Immunology and Infection

6.5K visualizações.  University of Kentucky. A mutagênese de troca de fitas cassadas é um método importante porque a exclusão genética é uma ferramenta valiosa que pode ser utilizada para entender a função dos produtos genéticos. A principal vantagem desta técnica é que ela permite a exclusão genética direcionada sem infligir efeitos polares em genes a jusante. Identifique aproximadamente três regiões quilobase diretamente rio acima e rio abaixo do gene destinado à exclusão para servir como os cinco braços de ecologia ...