I thank the George Church Lab for the kind gift of the pJA1 plasmid. I thank Fabienne Hamburger and Alex Boehm from the Urs Jenal Lab at the Biozentrum in Basel for help with bacterial conjugation and for providing the BW20767 strain. I also thank Olin Silander for helpful edits. Funding for this research was provided by funds from Massey University in New Zealand and the Swiss Initiative in Systems Biology (Project "Battle X" awarded to Dirk Bumann) at the University of Basel, Switzerland.

CAUTION: If using S. flexneri as a recipient strain, please note that S. flexneri is a human pathogen that can cause gastrointestinal disease. Experiments involving S. flexneri must be performed with appropriate biosafety precautions (designated BSL-2 in Europe and USA or PC2 in New Zealand). All experiments performed in the video associated with this protocol were done using the well-characterized recipient strain of non-pathogenic E. coli MG1655 in a PC1 setting. Work with Shigella flexneri was previously performed in a BSL-2 lab at the Biozentrum in Basel, as described in6.
Note: The following experiment is effectively done twice. The first part (Step 1.1- Step 4.5) is performed to find the best dilution for plating out the library, where the goal is to find a dilution of the conjugation which gives many individual colonies per plate, but not so many that a lawn forms. This is to reduce competition between mutants with significantly reduced fitness and those with more robust fitness. The second part (Steps 5.1- 7.4) is the creation of the final library, where many plates are spread of the appropriate dilution of the library.
1. To Prepare One Day Prior to Experiment
<ol>
	<li>Inoculate, from a frozen stock, the donor strain in 2 mL of LB broth, Millers recipe (NaCl at 10 g/L) media containing ampicillin at 100 &#181;g/mL. The donor strain used here is the E. coli strain BW2076715 harboring the pJA1 plasmid11. The use of ampicillin maintains selection for the pJA1 plasmid.</li>
	<li>Inoculate, from a single colony or frozen stock, the recipient strain in 2 mL of LB media with a resistance marker other than ampicillin or kanamycin. The recipient strain used here is the E. coli &#34;wild type&#34; strain MG165517,18 with a spontaneous resistance mutant to nalidixic acid. It is grown in 30 &#181;g/mL nalidixic acid.</li>
	<li>Prepare 20 Luria Broth containing 1.5% agar plates (in 90 mM petri dishes, approximately 12.5 mL). Agar plates lacking antibiotic can be stored at 4 &#176;C for several months until use.</li>
	<li>Prepare 20 Luria Broth containing 1.5% agar plates (in 90 mM petri dishes, approximately 12.5 mL) containing both nalidixic acid at 30 &#181;g/mL and kanamycin at 50 &#181;g/mL (LBA Nal30 Kan50). Agar plates containing antibiotics can be stored in the dark at 4 &#176;C for several months until use.</li>
	<li>Prepare 200 mL of sterile LB media. LB media can be stored at room temperature for several months.</li>
	<li>Prepare 1 mL of 100 mM sterile stock of IPTG, according to manufacturer&#39;s instructions.</li>
</ol>
2. Bacterial Mating or Conjugation
<ol>
	<li>Centrifuge 1 mL of overnight culture of the donor strain for 1 minute at 14,000 x g at room temperature. Discard growth media. This step is done to remove all growth media containing antibiotics.</li>
	<li>Resuspend the cell pellet into 110 &#181;L of LB (not containing antibiotics), thus concentrating the culture.</li>
	<li>Using sterile forceps, position a sterile nitrocellulose filter (alternatively a sterile piece of blotting paper can be used, see Materials list) onto the middle of an LBA plate (not containing antibiotics). Label the plate &#34;Negative Control: Donor&#34;.</li>
	<li>Using a pipette, drop 50 &#181;L of the concentrated donor culture onto the filter.</li>
	<li>Repeat steps 2.1-4 for the recipient strain, labeling the plate &#34;Negative Control: Recipient.&#34;</li>
	<li>For the library, drop 50 &#181;L of the donor (from step 2.2) AND 50 &#181;L of the recipient strain (from step 2.5) onto a single sterile filter on an LBA plate (these plates should not contain antibiotics). Label this plate &#34;Library.&#34; Mating occurs because the donor and recipient are in close physical contact on the filter.</li>
	<li>Place the agar plates containing the filters at 37 &#176;C for 6 h.</li>
</ol>
3. Activation of pJA1 Transposon by IPTG Induction of the Transposase
<ol>
	<li>Prepare three 15 mL conical vials containing 2 mL of LB with IPTG at a final concentration of 1 mM (i.e., 20 &#181;L of 100 mM stock of IPTG). Label each: Donor Control, Recipient control, and Library.</li>
	<li>Remove the plates from the incubator after 6 h of growth at 37 &#176;C (from step 2.4). Some bacterial growth should be visible on the filter.</li>
	<li>Using sterile forceps, remove the filter paper from the donor control and place it in the 15 mL conical vial labeled Donor Control (from step 3.1). Do the same for the Recipient Control and Library.</li>
	<li>Tap the tubes to get the filter paper to the bottom of the 15 mL conical vial and ensure that it is fully submerged in LB.</li>
	<li>Vortex tubes for at least a full minute on high to disassociate the bacteria from the nitrocellulose filter. The LB should become cloudy with bacterial cells (Figure 2).</li>
	<li>Using sterile glass beads or a sterile spreader, plate 200 &#181;L of the vortexed bacteria suspension from the donor control onto LBA Nal30 Kan50 plates labeled &#34;Donor Control, 200 &#181;L&#34;.</li>
	<li>Repeat the step above (3.6) for the recipient control, labeling the plates &#34;Recipient Control, 200 &#181;L&#34;.</li>
	<li>Repeat the step above (3.7) for the library, labeling the plates &#34;Library, 200 &#181;L&#34;.</li>
	<li>Make additional dilutions (i.e., 1:5, 1:10 and 1:100 dilutions, using LB not containing antibiotics as a diluent) of the library. Plate 200 &#181;l of undiluted library and additional dilutions onto appropriately labeled LBA Nal30 Kan50 plates.</li>
	<li>Place plates into the 37 &#176;C incubator for 18 h. Clones with transposons inserted in a gene that causes a large loss of fitness may take longer to grow and appear on the plate. There should be a variety of colony sizes (Figure 3). Donor and recipient strain plates should have no colonies on them.</li>
</ol>
4. Choose the Appropriate Dilution of the Library to be Used for the Final Mutant Library
<ol>
	<li>Determine a dilution of the library from Step 3.9 that yields many colonies of varying sizes that are adequately spaced. The aim is to get as many colonies as possible on one plate, but not so many that the colonies merge into one another and compete for resources. This number should be approximately 500-2,000 colonies per plate. An example of appropriate colony densities on a plate is shown in Figure 3.</li>
	<li>Record the number of colonies on the plates.</li>
	<li>Calculate the frequency of conjugation (the number of conjugants per recipient cell). This should be in the range of 1 x 10-4 to 1 x 10-6 19.</li>
	<li>Determine the number of mutants desired in the final library based on downstream applications. Many users simply require a library with as many mutants as possible. To generate as many transposon insertions as theoretically possible (one insertion every base pair), aim for approximately the same number of colonies as base pairs in the genome (i.e., ~4.5 x 106 for E. coli) to fully saturate the genome with all possible transposon insertions. It is important to note, many mutants harboring insertions in essential or functionally important genes will not be able to be recovered, as these mutations will be fatal to the recipient.</li>
	<li>Calculate how much volume of the initial library is needed to create a library of the desired size. For example, the desired library size is 5 x 104 mutants. The dilution with the best spacing of colonies from Step 3.9 was 200 &#181;L of a 1:10 dilution. The number of colonies on this plate is estimated to be approximately 2,000 colonies. If 5 x 104 mutants are needed and each plate has 2,000 colonies, then 25 plates will be needed at this dilution.</li>
</ol>
5. Creation of the Final Mutant Library
<ol>
	<li>Repeat steps 1 to 3.8, adjusting the number of plates at step 1.4 as needed (see step 4.4).</li>
	<li>At step 3.9, plate the dilution chosen in step 4.1 onto as many plates as needed to create the library of the desired size (see step 4.4).</li>
</ol>
6. Estimate Library Density
<ol>
	<li>Count how many colonies there are per plate, and thereby get a rough estimate of how dense the library is. For example: A total of 2x105 colonies are plated. The E. coli MG1655 genome is about 4.5 x 106 base pairs. Therefore, a library with about 2 x 105 inserts means that there is an insert on average every 22 base pairs and that each gene should be mutated about 45 times, given that one gene is approximately 1 x 103 base pairs. Insertions in essential genes are likely to be very underrepresented in this library, and thus the density in non-essential genes is likely to be greater than this. This sort of calculation provides one with a broad estimate of transposon density.</li>
</ol>
7. Pooling Transposon Library and Storage
<ol>
	<li>After counting colonies, add 1 mL of LB (or more, as needed) to the library plate and use a sterile spreader to scrape off bacteria from the plate. Remove the bacterial suspension and place in a 50 mL or 15 mL conical vial. Repeat for all plates.</li>
	<li>Vortex the pooled bacteria suspension for a full minute to homogenize the suspension.</li>
	<li>Add sterile glycerol to a final concentration of 20%.
	<ol>
		<li>Prepare 100 x 20 &#181;L aliquots in 0.25 mL tubes for easy re-growth.</li>
		<li>Prepare at least two or more 1 mL aliquot in cryovials.</li>
	</ol>
	</li>
	<li>Store all aliquots at -80&#176;C</li>
</ol>

<ol>
	<li>Camacho, L. R., Ensergueix, D., Perez, E., Gicquel, B., Guilhot, C. <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+virulence+gene+cluster+of+Mycobacterium+tuberculosis+by+signature-tagged+transposon+mutagenesis.">Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis.</a> Mol Microbiol. 34 (2), 257-267 (1999).</li><li>Hensel, M., Shea, J. E., Gleeson, C., Jones, M. D., Dalton, E., Holden, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+identification+of+bacterial+virulence+genes+by+negative+selection.">Simultaneous identification of bacterial virulence genes by negative selection.</a> Science. 269 (5222), 400-403 (1995).</li><li>Glass, J. I., Assad-Garcia, N., 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=Essential+genes+of+a+minimal+bacterium.">Essential genes of a minimal bacterium.</a> PNAS. 103 (2), 425-430 (2006).</li><li>Salama, N. R., Shepherd, B., Falkow, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+transposon+mutagenesis+and+essential+gene+analysis+of+Helicobacter+pylori.">Global transposon mutagenesis and essential gene analysis of Helicobacter pylori.</a> J Bacteriol. 186 (23), 7926-7935 (2004).</li><li>Akerley, B. J., Rubin, E. J., Camilli, A., Lampe, D. J., Robertson, H. M., Mekalanos, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+identification+of+essential+genes+by+in+vitro+mariner+mutagenesis.">Systematic identification of essential genes by in vitro mariner mutagenesis.</a> PNAS. 95 (15), 8927-8932 (1998).</li><li>Freed, N. E., Bumann, D., Silander, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+Shigella+Tn-seq+data+with+gold-standard+E.+coli+gene+deletion+data+suggests+rare+transitions+between+essential+and+non-essential+gene+functionality.">Combining Shigella Tn-seq data with gold-standard E. coli gene deletion data suggests rare transitions between essential and non-essential gene functionality.</a> BMC Microbiology. 16 (1), 1-14 (2016).</li><li>van Opijnen, T., Bodi, K. L., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tn-seq:+high-throughput+parallel+sequencing+for+fitness+and+genetic+interaction+studies+in+microorganisms.">Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms.</a> Nature Methods. 6 (10), 767-772 (2009).</li><li>Langridge, G. C., Phan, M. 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=Simultaneous+assay+of+every+Salmonella+Typhi+gene+using+one+million+transposon+mutants.">Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants.</a> Genome Research. 19 (12), 2308-2316 (2009).</li><li>Gawronski, J. D., Wong, S. M. S., Giannoukos, G., Ward, D. V., Akerley, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+insertion+mutants+within+libraries+by+deep+sequencing+and+a+genome-wide+screen+for+Haemophilus+genes+required+in+the+lung.">Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung.</a> PNAS. 106 (38), 16422-16427 (2009).</li><li>Chan, K., Kim, C. C., Falkow, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+detection+of+Salmonella+enterica+serovar+Typhimurium+transposon+mutants+that+cannot+survive+in+macrophages+and+mice.">Microarray-based detection of Salmonella enterica serovar Typhimurium transposon mutants that cannot survive in macrophages and mice.</a> Infect and Immun. 73 (9), 5438-5449 (2005).</li><li>Badarinarayana, V., Estep, P. W., 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=Selection+analyses+of+insertional+mutants+using+subgenic-resolution+arrays.">Selection analyses of insertional mutants using subgenic-resolution arrays.</a> Nature biotechnol. 19 (11), 1060-1065 (2001).</li><li>Bender, J., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IS10+transposase+mutations+that+specifically+alter+target+site+recognition.">IS10 transposase mutations that specifically alter target site recognition.</a> The EMBO Journal. 11 (2), 741-750 (1992).</li><li>Shafferman, A., Helinski, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+properties+of+the+beta+origin+of+replication+of+plasmid+R6K.">Structural properties of the beta origin of replication of plasmid R6K.</a> J Biol Chem. 258 (7), 4083-4090 (1983).</li><li>Langridge, G. C., Phan, M. 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=Simultaneous+assay+of+every+Salmonella+Typhi+gene+using+one+million+transposon+mutants.">Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants.</a> Genome Research. 19 (12), 2308-2316 (2009).</li><li>Metcalf, W. W., Jiang, W., Daniels, L. L., Kim, S. K., Haldimann, A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditionally+replicative+and+conjugative+plasmids+carrying+lacZ+alpha+for+cloning,+mutagenesis,+and+allele+replacement+in+bacteria.">Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria.</a> Plasmid. 35 (1), 1-13 (1996).</li><li>Badarinarayana, V., Estep, P. W., 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=Selection+analyses+of+insertional+mutants+using+subgenic-resolution+arrays.">Selection analyses of insertional mutants using subgenic-resolution arrays.</a> Nature biotechnol. 19 (11), 1060-1065 (2001).</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+&#947;&#948;.">Identification of a Sex-factor-affinity Site in E. coli as &#947;&#948;.</a> Cold Spring Harbor Symposia on Quantitative Biology. 45, 135-140 (1981).</li><li>Blattner, F. R., Plunkett, G., 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=The+Complete+Genome+Sequence+of+Escherichia+coli+K-12.">The Complete Genome Sequence of Escherichia coli K-12.</a> Science. 277 (5331), 1453-1462 (1997).</li><li>Alexeyev, M. F., Shokolenko, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-Tn10+transposon+derivatives+for+insertion+mutagenesis+and+gene+delivery+into+the+chromosome+of+gram-negative+bacteria.">Mini-Tn10 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of gram-negative bacteria.</a> Gene. 160 (1), 59-62 (1995).</li><li>Kvitko, B. H., Bruckbauer, S., 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=A+simple+method+for+construction+of+pir++Enterobacterial+hosts+for+maintenance+of+R6K+replicon+plasmids.">A simple method for construction of pir+ Enterobacterial hosts for maintenance of R6K replicon plasmids.</a> BMC Res Notes. 5 (1), 157 (2012).</li><li>Barrick, J. E., Yu, D. S., 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=Genome+evolution+and+adaptation+in+a+long-term+experiment+with+Escherichia+coli.">Genome evolution and adaptation in a long-term experiment with Escherichia coli.</a> Nature. 461 (7268), 1243-1247 (2009).</li><li>Jacobs, M. A., Alwood, 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=Comprehensive+transposon+mutant+library+of+Pseudomonas+aeruginosa.">Comprehensive transposon mutant library of Pseudomonas aeruginosa.</a> PNAS. 100 (24), 14339-14344 (2003).</li><li>van Opijnen, T., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon+insertion+sequencing:+a+new+tool+for+systems-level+analysis+of+microorganisms.">Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms.</a> Nature Rev Microbiol. 11 (7), 435-442 (2013).</li></ol>

The author has nothing to disclose.

The protocol described here allows for the construction of a dense insertion library. This method allows for the creation of a transposon library with over 2 x 105 unique transposon mutants using under 5 mL of culture volume6. It is relatively easy to perform, uses reagents available in most basic microbiology labs, is scalable, and requires little in the way of expensive equipment or consumables such as electroporation cuvettes.
A significant benefit of this method is that, in theory, the user has wide latitude in the choice of enterobacterial recipient strains. This paper, as well as others11, use E. coli as a recipient strain, however the pJA1 plasmid has been used successfully with other enterobacterial recipient species such as Shigella flexneri6 and Salmonella enterica serovar Typhimurium strain SL134410. Theoretically, the &#947; origin of replication (oriR6K&#947;) in pJA1 allows this plasmid to be maintained in a broad host range19, allowing that the recipient strain is pir+. Recently, new methods have been described that allow for construction of the pir+ in a range of enterobacterial strains20, giving additional flexibility. Additionally, the300 base pair mob region from the RP4 plasmid in pJA1 allows conjugative transfer of this plasmid to a wide range of gram negative bacterial strains19. Simply put, this method could theoretically be used with a variety of recipient strains, as long as several conditions are met: the strain is pir+, and is marked with an antibiotic resistance other than kanamycin and other than the donor strain.
A critical step in the protocol lies in estimating the proper number of cells to plate out in step 4.1. If colonies are spaced too closely, they compete for nutrients on the plate and less fit mutants are outcompeted. This may lead to a reduction in the total number of mutants. Alternatively, if colonies are spaced too far apart, there will be too few colonies on the plate, and the total number of agar plates needed to achieve a large library becomes burdensome. Therefore, achieving the right balance in terms of colony numbers per plate is important.
It is important to perform the controls listed in the protocol to ensure the steps are working as described. Notably, when using nalidixic acid as a counter-selection against the donor strain, it is important to ensure the negative donor control plates are free of colonies. This is because there may be a low rate (approximately 1 x 10-10)21 of spontaneous resistance to nalidixic acid, yielding false positives. Typically, the rate of conjugation and transposition is approximately 2 x 10-4 19. Therefore, the rate of conjugation and transposition is several orders of magnitude greater than the rate of spontaneous resistance to nalidixic acid. Therefore, the rate of false positives compared to true transposition events is very low and deemed negligible when the protocol is working. However, if rates of conjugation or transposition are significantly reduced, (from low mating efficiency and/or lack of induction of the transposase gene with IPTG) and the protocol is scaled up to compensate for this, then the number of false positives (clones that do not have the transposon inserted) may also increase.
Some modifications can be made to incubation times in Steps 3.2 and 3.10. Step 3.2 states that conjugation should occur for 6 h, but in our experience, this time step can be varied (i.e., 4-7 h) without changing the results significantly. Additionally, in step 3.10, the length of time the colonies are incubated on the agar plates can also be adjusted. This can be varied depending on the average doubling time or growth rate of the recipient strain. Additionally, in our experience, 18 h yielded a variety of colony sizes, indicating a library of diverse fitness. However, colonies with greatly reduced fitness may take longer to grow and thus may not be visible after 18 h. If this method is being used to find clones of extremely reduced fitness, longer incubation times and fewer colonies on the plate to reduce crowding (i.e., 48 h, 50-300 colonies) may be used.
Additional pitfalls of this method include that it is not possible to use a recipient strain that is already kanamycin resistant. It may be possible to swap out the kanamycin resistance marker in the pJA1 plasmid for an alternative selectable marker, such as chloramphenicol resistance to overcome this hurdle. It may also be worth noting that, in theory, it is possible to use a recipient strain that is ampicillin resistant, as the pJA1 plasmid containing the ampicillin resistance marker is lost shortly after transposition.
The creation of a dense transposon library in the genetic background of choice is potentially advantageous for many downstream applications. For example, a dense transposon library could be used to identify auxotrophic mutants using replica plating22 or to identify mutants that are defective in establishing an infection1,2. More recently, as DNA sequencing costs have dropped and new technologies such as next generation sequencing have become commonplace, transposon libraries have been used with deep DNA sequencing to gain insight into gene essentiality, gene function, and genetic interactions. Some of these methods are reviewed in 23 and include methods such as transposon-directed insertion site sequencing (TraDIS), transposon sequencing (Tn-seq), high-throughput insertion tracking by deep sequencing (HITS), and insertion sequencing (INSeq). All these downstream methods rely on the construction of dense transposon insertion libraries. While other vectors may need to be used for particular downstream methods, the protocol described here gives an overview of the salient procedural points to follow.

An erratum was issued for: <a href="https://jove.unicartagenaproxy.elogim.com/video/56216/creation-dense-transposon-insertion-library-using-bacterial">Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri</a>. The Discussion and References sections have been corrected.
The second paragraph in the Discussion section was updated from:
A significant benefit of this method is that, in theory, the user has wide latitude in the choice of enterobacterial recipient strains. This paper, as well as others11, use&#160;E. coli&#160;as a recipient strain, however the pJA1 plasmid has been used successfully with other enterobacterial recipient species such as&#160;Shigella flexneri6&#160;and&#160;Salmonella enterica&#160;serovar Typhimurium strain SL134410. Theoretically, the &#947; origin of replication (oriR6K&#947;) in pJA1 allows this plasmid to be maintained in a broad host range19, allowing that the recipient strain is&#160;pir+.&#160;Recently, new methods have been described that allow for construction of the&#160;pir+&#160;in a range of enterobacterial strains20, giving additional flexibility. Additionally, the300 base pair mob&#160;region from the RP4 plasmid in pJA1 allows conjugative transfer of this plasmid to a wide range of gram negative bacterial strains19. Simply put, this method could theoretically be used with a variety of recipient strains, as long as several conditions are met: the strain is&#160;pir+, and is marked with an antibiotic resistance other than kanamycin and other than the donor strain.
to:
A significant benefit of this method is that, in theory, the user has wide latitude in the choice of enterobacterial recipient strains. This paper, as well as others11, use&#160;E. coli&#160;as a recipient strain, however the pJA1 plasmid has been used successfully with other enterobacterial recipient species such as&#160;Shigella flexneri6&#160;and&#160;Salmonella enterica&#160;serovar Typhimurium strain SL134410. Theoretically, the &#947; origin of replication (oriR6K&#947;) in pJA1 allows this plasmid to be maintained in a broad host range19, allowing that the recipient strain is&#160;pir-.&#160;The&#160;300 base pair mob&#160;region from the RP4 plasmid in pJA1 allows conjugative transfer of this plasmid to a wide range of gram negative bacterial strains19. Simply put, this method could theoretically be used with a variety of recipient strains, as long as several conditions are met: the strain is&#160;pir-, and is marked with an antibiotic resistance other than kanamycin and other than the donor strain.
The References&#160;section was updated from:
<ol start="18">
	<li>Blattner, F. R., Plunkett, G.,&#160;et al.&#160;The Complete Genome Sequence of Escherichia coli K-12.&#160;Science.&#160;277&#160;(5331), 1453-1462 (1997).</li>
	<li>Alexeyev, M. F., &#38; Shokolenko, I. N. Mini-Tn10 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of gram-negative bacteria.&#160;Gene.&#160;160&#160;(1), 59-62 (1995).</li>
	<li>Kvitko, B. H., Bruckbauer, S.,&#160;et al.&#160;A simple method for construction of pir+ Enterobacterial hosts for maintenance of R6K replicon plasmids.&#160;BMC Res Notes.&#160;5&#160;(1), 157 (2012).</li>
	<li>Barrick, J. E., Yu, D. S.,&#160;et al.&#160;Genome evolution and adaptation in a long-term experiment with Escherichia coli.&#160;Nature.&#160;461&#160;(7268), 1243-1247 (2009).</li>
	<li>Jacobs, M. A., Alwood, A.,&#160;et al.&#160;Comprehensive transposon mutant library of Pseudomonas aeruginosa.&#160;PNAS.&#160;100&#160;(24), 14339-14344 (2003).</li>
	<li>van Opijnen, T., &#38; Camilli, A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms.&#160;Nature Rev Microbiol.&#160;11&#160;(7), 435-442 (2013).</li>
</ol>
to:
<ol start="18">
	<li>Blattner, F. R., Plunkett, G.,&#160;et al.&#160;The Complete Genome Sequence of Escherichia coli K-12.&#160;Science.&#160;277&#160;(5331), 1453-1462 (1997).</li>
	<li>Alexeyev, M. F., &#38; Shokolenko, I. N. Mini-Tn10 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of gram-negative bacteria.&#160;Gene.&#160;160&#160;(1), 59-62 (1995).</li>
	<li>Barrick, J. E., Yu, D. S.,&#160;et al.&#160;Genome evolution and adaptation in a long-term experiment with Escherichia coli.&#160;Nature.&#160;461&#160;(7268), 1243-1247 (2009).</li>
	<li>Jacobs, M. A., Alwood, A.,&#160;et al.&#160;Comprehensive transposon mutant library of Pseudomonas aeruginosa.&#160;PNAS.&#160;100&#160;(24), 14339-14344 (2003).</li>
	<li>van Opijnen, T., &#38; Camilli, A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms.&#160;Nature Rev Microbiol.&#160;11&#160;(7), 435-442 (2013).</li>
</ol>

An erratum was issued for: <a href="https://jove.unicartagenaproxy.elogim.com/video/56216/creation-dense-transposon-insertion-library-using-bacterial">Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri</a>.&#160;The Discussion and References sections have been corrected.

Erratum: Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

The creation of transposon mutagenesis libraries in bacteria is useful for a wide variety of applications, ranging from discoveries of virulence genes in bacterial pathogens1,2, to studies of essential genes3,4,5,6, to the identification of genetic interaction networks7,8,9. Critical to these studies is the ability to create a large library of mutants. The use of transposons (short fragments of DNA that insert randomly into a genome) is a simple means of disrupting gene function, as the insertion of a transposon within the open reading frame or regulatory region of a gene will often disrupt the function or expression of the gene.
Outlined here is a method for the creation of a transposon library in either E. coli or S. flexneri by bacterial conjugation using the plasmid pJA110. There are two main advantages to using this plasmid. The first advantage is that the variant of the Tn10 transposase expressed from the pJA1 plasmid is inducible and has low insertion bias11,12, meaning that the transposon will randomly integrate into the genome when Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) is added to the media. The transposon contains a kanamycin resistance marker, allowing for the selection of mutants with the transposon inserts into the chromosome of the recipient strain. The second advantage of the pJA1 plasmid is that it contains an R6K mutant origin of replication. The R6K mutant origin of replication requires the lambda pir gene in order for the maintenance of the plasmid13. As the plasmid cannot replicate in pir- strains, it will be lost in the recipient strain (Figure 1). This ensures that Tn10 transposase is removed from the cell and is no longer active, reducing further mutations after the initial transposition event.
The use of bacterial conjugation to move the pJA1 plasmid from the donor strain to the recipient strain is advantageous for several reasons. Conjugation is simple and inexpensive to perform and does not need special equipment such as an electroporator. Additionally, the high efficiency of bacterial conjugation allows for a very large library (&#62;2 x 105 unique insertions) to be achieved with just a few milliliters (mL) of overnight bacterial culture6. The process takes around two hours of hands-on time along with time for incubation and growth of the bacteria. Langridge et al.14 reported performing 130 electroporations to create a transposon mutagenesis library of a size similar to the one described here8, which is achieved with a single conjugation. The use of 130 electroporations requires labor intensive and time-consuming preparation of electrocompetent cells and the use of many expensive materials (e.g., electroporation cuvettes), at a cost of more than $1,000 USD in consumables alone. Other studies10 have used similar methods, but with different bacterial strains, and achieved far smaller library sizes (5 x 104 colony forming units) than reported here.
Notes on the donor strain: The donor strain used here is the E. coli strain BW2076715 containing the pJA1 transposon plasmid16. Strain BW20767 can conjugate to other strains, making the transfer of the pJA1 plasmid highly efficient. Also, importantly, BW20767 is lambda pir+. As mentioned previously, the plasmid pJA1 is only able to be maintained in strains containing the lambda pir gene. This strain is kanamycin and ampicillin resistant. The pJA1 plasmid contains the ampicillin resistance marker, and a kanamycin resistance marker is contained within the transposon. This strain is grown with selection on the plasmid using ampicillin at 100 &#181;g/mL. It is also worth noting that other strains harboring constitutively expressed transposase genes are known to be unstable, and while the transposase used here is under inducible control, there remains a low risk of leaky expression. For this reason, it is suggested that the passage of this strain should be minimized and a fresh streak should be taken from a frozen culture for each new library preparation. The donor strain used here is available from our lab upon request.
Notes on the Recipient strain: The recipient strain can be a strain of choice, such as K12-derived lab strains of E. coli or strains of S.flexneri (also, see Discussion). The recipient strain must have an additional antibiotic resistance marker that is not kanamycin resistance, so that the donor strain can be selected against. For the recipient strain, an E. coli MG1655 strain is used here with an introduced spontaneous nalidixic acid mutation. The spontaneous nalidixic acid mutant was selected by plating out 2 mL of overnight culture in 200 &#181;L aliquots onto plates containing nalidixic acid at 30 &#181;g/mL. A single clone of MG1655 was selected that was resistant to nalidixic acid to become the recipient strain. Additionally, the recipient strain must be lambda pir negative, as described above.
Overview: Once bacterial conjugation has taken place and the pJA1 plasmid has moved from the donor strain into the recipient strain, the addition of IPTG to the media will induce the expression of the tnp gene, which is under control of the IPTG-inducible lacIq/Ptac promoter (Figure 1). The tnp gene on pJA1 is a mutant transposase which has a lower frequency of insertion at hot spots6,10,11. Upon addition and induction with IPTG, the transposon is activated and randomly inserted in the genome. The plasmid cannot be maintained in the lambda pir- recipient strain and is lost.

<table><tbody><tr><td>0.45 micron nitrocellulose filters (sterile)</td><td>Millipore</td><td>HAWP02500</td><td>Alternatively blotting paper can be used (listed below)</td></tr><tr><td>Blotting paper cut into ~ 1 inch circles, then autoclave/sterilized in foil. There is no difference in efficiency between the nitrocellulose filters or blotting paper</td><td>GE life Sciences: product Grade 3MM Chr Blotting Paper, sheet, 46 &amp;times; 57 cm</td><td>3030-917</td><td>Alternatively nitrocellose filters can be used (listed above)</td></tr><tr><td>Metal Forceps, sterile</td><td>VWR/Global Science New Zealand</td><td>MURRE009/01</td><td></td></tr><tr><td>Petri dishes, sterile, 90 mm diameter, 68 mL volume,&amp;nbsp; 12.5 mL working volume.</td><td>Interlab New Zealand</td><td>2303-1090</td><td></td></tr><tr><td>Sterile glass spreaders</td><td>VWR/Global Science New Zealand</td><td>NZNZSPREADER</td><td>Alternatively sterile glass beads can be used for spreading culture onto plates (listed below)</td></tr><tr><td>Sterile glass beads</td><td>VWR/Global Science New Zealand</td><td>HERE1080603</td><td>Alternatively a sterile glass spreader can be used for spreading culture onto plates (listed above)</td></tr><tr><td>Nalidixic acid (optional)</td><td>GoldBio.com</td><td>N-015-250</td><td></td></tr><tr><td>Ampicillin sodium salt BioChemica</td><td>VWR/Global Science New Zealand</td><td>APLIA0839.0025</td><td>prepare according to manufacturer recommendation</td></tr><tr><td>Kanamycin sulfate BioChemica</td><td>VWR/Global Science New Zealand</td><td>APLIA1493.0010</td><td></td></tr><tr><td>IPTG (Isopropyl &amp;beta;-D-1-thiogalactopyranoside)&amp;nbsp;</td><td>Thermofisher New Zealand</td><td>FMTR0393</td><td></td></tr><tr><td>Difco Agar granulated</td><td>Fort Richard Laboratries/Interlab New Zealand</td><td>214530</td><td></td></tr><tr><td>Luria Broth Base (Miller&#039;s LB Broth Base)</td><td>Thermofisher</td><td>12795-084</td><td></td></tr><tr><td>Glycerol bidistilled 99,5 %</td><td>VWR/Global Science New Zealand</td><td>VWRC24388.320</td><td></td></tr><tr><td>15 ml polypropelene sterile conical vials&amp;nbsp;</td><td>VWR/Global Science New Zealand</td><td>CORN430791</td><td></td></tr><tr><td>Microcentrifuge tubes, flat cap, 1.7ml, Ultra-clear PP, graduated, autoclavable and freezable</td><td>VWR/Global Science New Zealand</td><td>AXYGMCT-175-C</td><td></td></tr><tr><td>50ml Centrifuge tubes, Falcon, PP, conical, printed graduation, with flat screw caps, sterile</td><td>VWR/Global Science New Zealand</td><td>BDAA352070</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Incubator at 37C</td><td></td><td></td><td></td></tr><tr><td>Autoclave for sterilizing media&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

creation of a dense transposon insertion library using bacterial conjugation in enterobacterial strains such as escherichia coli or shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

After 18 hours of growth, plates should contain many colonies with a variety of colony sizes (Figure 3). Different colony sizes indicate clones of varying fitness, and are a good sign that the protocol has worked. The donor and recipient controls should have no growth on the plates. Using the protocol above should yield well over 2 x 105 colony forming units (CFU). Scaling up the protocol to five replicate conjugations at once should yield approximately 1 x 106 CFU. In previous work, analysis using next generation sequencing indicated that such a scaled up library should yield &#62;2 x 105 unique insertions6. At very high CFU (i.e., 1 x 106 CFU) the rate of unique insertions is expected to plateau, as all available (non-fatal) insertions become represented.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56216/56216fig1.jpg" /> 
Figure 1: Schematic of bacterial conjugation and insertion of transposon into the chromosome. <a href="https://cloudflare.jove.com/files/ftp_upload/56216/56216fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56216/56216fig2.jpg" /> 
Figure 2: Image of filters submersed in LB media before and after vortexing. Before vortexing, cells are stuck to the filter and the media is clear. After vortexing, the media becomes cloudy as cells have come off of the filter and are in the media. <a href="https://cloudflare.jove.com/files/ftp_upload/56216/56216fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56216/56216fig3.jpg" /> 
Figure 3: (A) Representative plate of transposon library after 18 h of growth. (B) Close up of colony sizes. <a href="https://cloudflare.jove.com/files/ftp_upload/56216/56216fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri at JoVE.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

creation-dense-transposon-insertion-library-using-bacterial

Research

JoVE Journal

Immunology and Infection

15.8K Views.  Massey University. Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Video: Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

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.

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 ...

Genetics

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Bioengineering

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2&#8211;0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

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.

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 ...

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the study of the mobilization of naturally occurring conjugative plasmids, using two naturally-occurring plasmids as an example. These protocols rely on the differential presence of selectable markers in donor, recipient, and conjugative plasmid. Specifically, the methods described include 1) the identification of natural conjugative plasmids, 2) the quantification of conjugation rates in solid culture, and 3) the diagnostic detection of the antibiotic resistance genes and plasmid replicon types in transconjugant recipients by polymerase chain reaction (PCR). The protocols described here have been developed in the context of studying the evolutionary ecology of horizontal gene transfer, to screen for the presence of conjugative plasmids carrying antibiotic-resistance genes in bacteria found in the environment. The efficient transfer of conjugative plasmids observed in these experiments in culture highlights the biological relevance of conjugation as a mechanism promoting horizontal gene transfer in general and the spread of antibiotic resistance in particular.

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation mediates horizontal gene transfer by mobilizing plasmid DNA across two different cells, facilitating the spread of beneficial genes. This work describes a widely-used method for the efficient detection of conjugative plasmid transfer, based on the use of differential markers in the conjugative plasmid, donor, and recipient to detect transconjugation.

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

Biology

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The &#8220;last-line&#8221; antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways ...

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identify&#160;molecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts.
Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette is&#160;carried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

This is an adapted method for identifying candidate insect colonization factors in a Burkholderia beneficial symbiont. The beetle host is infected with a random mutant library generated via transposon mutagenesis, and library complexity after colonization is compared to a control grown in vitro.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

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Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles