The author would like to thank all of my undergraduate Independent Research Students and all of my Microbial Physiology graduate students who tested my transposon mutagenesis ideas during the 2010-2014 spring semesters. This work was funded by the Department of Biological Sciences at Youngstown State University.

1. Electroporation of Competent Cells5,11
<ol>
	<li>Dilute an O/N LB culture of Enterobacter sp. YSU 1/20 into 50 ml of fresh LB medium and grow with shaking at 120 rpm and 30 &#176;C to an O.D. (600 nm) between 0.4 and 0.6. Optionally, grow other bacterial strains at their optimal growth temperature.</li>
	<li>Chill the cells on ice for 5 min and centrifuge at 4 &#176;C and 7,000 x g for 5 min.</li>
	<li>Discard the supernatant, resuspend the cells in 50 ml of sterile ice cold water and centrifuge at 4 &#176;C and 7,000 x g for 5 min. Repeat this step.</li>
	<li>Discard the supernatant and resuspend the cells in a volume of ice cold water equal to the cell pellet volume. For long term storage, wash the cells with 10 ml of ice cold 10% (v/v) glycerol, resuspend in an equal pellet volume of ice cold 10% (v/v) glycerol, store at -80 &#176;C, and thaw on ice before electroporation.</li>
	<li>Add 0.5 &#181;l of the transposome to 40 &#181;l of cells. The commercially available transposome solution is supplied at a DNA concentration of 0.33 &#181;g/&#181;l. Although 0.33 &#181;g is recommended, 0.165 &#181;g DNA is sufficient.</li>
	<li>Place the cell/transposome mixture in an ice cold, 0.2 mm, electroporation cuvette and tap the mixture to the bottom of the cuvette. Shock the cells at 25 &#181;F, 200 &#937;, and 2.5 kV. To ensure that the cells remain chilled, store the electroporation cuvettes in a -20&#176;C freezer before use.</li>
	<li>Immediately, add 960 &#181;l of filter sterilized super optimal broth with catabolite repression (SOC) medium. Mix by pipetting up and down and transfer the cells to a sterile 1.5 ml microfuge tube. 
	NOTE: The cells begin to die after the shock, but the salt in the SOC medium allows them to recover. To economize, it is not necessary to add transposome or to shock the negative control cells. Just add 960 &#181;l of sterile SOC medium to it.</li>
	<li>Incubate the cells at 30 &#176;C for 45-60 min with shaking at 120 rpm. This provides time for the transposome to recombine with the host genome and for the cells to express kanamycin resistance.</li>
	<li>Spread 100 &#181;l of cells on a LB-kan agar plate and incubate the plate O/N&#160;at 30 &#176;C. Store the remaining transformation mix in the refrigerator at 4 &#176;C. Spread additional amounts at a later date up to 2 weeks after the initial electroporation if required.</li>
</ol>
2. Gridding of Transformants
<ol>
	<li>Tape a grid to the lid of an empty 100 x 15 mm petri dish. Copy a grid from &#8220;A Short Course in Bacterial Genetics&#8221;12.</li>
	<li>Draw a line on the bottom of an LB-kan agar plate. Use a small piece of tape to anchor it to the gridded lid so that the grid is visible through the agar. Ensure that the line on the bottom of the plate is aligned with the top, center of the grid. Anchoring the plate with tape keeps it from sliding, allowing for even gridding. The line facilitates colony identification after replica plating.</li>
	<li>Pick a single transformant with a sterile toothpick and spot it in the center of a square in the grid. Just touch the colony and the spot. Do not dig the toothpick into the agar. To avoid contaminating the plate, handle the toothpick only at one end.</li>
	<li>Spot another transformant in an adjacent square as in step 2.3. When the plate is full, incubate it O/N at 30 &#176;C. This is the master plate.</li>
</ol>
3. Replica Plating to Determine the Auxotroph Phenotype12
<ol>
	<li>For each plate that was gridded, make a stack of three fresh agar plates numbered one through three: one for LB-kan, two for M-9-kan and three for LB-kan. Dry the plates upside down, O/N, at 37 &#176;C before use.</li>
	<li>Draw a line on the top of each plate as in step 2.2.</li>
	<li>Wipe the replica plating tool with 70% ethanol, place a sterile velveteen square on top of the replica plating tool and clamp the velveteen square down. Hands are teeming with microbes even after washing them. Prevent the introduction of contaminating microbes by handling the velveteen square by its edge.</li>
	<li>Align the line on the master plate with a line drawn on the clamp and place the plate on top of the velveteen square. Apply gentle pressure to transfer the bacteria from the plate to the velveteen square. Be careful not to smear the bacteria. Remove the master plate from the velveteen square.</li>
	<li>Align the line drawn on plate number one with the line on the clamp and place it on top of the velveteen square. Apply gentle pressure to transfer the bacteria from the velveteen square to the plate. Remove plate number one.</li>
	<li>Discard the velveteen square into a beaker of water and place a clean, sterile velveteen square on the replica plating tool as in step 3.3. This step prevents over-inoculation which causes breakthrough growth and false positive results.</li>
	<li>Align the line on plate number one with the line on the clamp and place it on top of the new velveteen square. Apply gentle pressure to transfer the bacteria from plate number one to the velveteen square. Remove plate number one.</li>
	<li>Align the line drawn on plate number two with the line on the clamp and place it on top of the velveteen square. Apply gentle pressure to transfer the bacteria from the velveteen square to plate number two. Remove plate number two.</li>
	<li>Repeat step 3.8 for plate number three. The third plate is a control for complete transfer from the master plate to the other two plates. Discard the velveteen square into a beaker of water. To reuse the velveteen squares, autoclave the beaker containing the contaminated velveteen squares, wash them, dry them, wrap them in aluminum foil and re-autoclave them.</li>
	<li>Incubate the plates at 30 &#176;C O/N. Colonies that grow on both LB-kan agar plates but fail to grow on M-9-kan plates are considered to be auxotrophs (Figure 2).</li>
	<li>Streak out auxotrophs from plate number one onto a fresh LB-kan agar plate, and incubate the plate at 30 &#176;C O/N.</li>
	<li>Streak out for isolation 3 colonies from the streak plate in step 3.11 onto a fresh LB-kan plate. Incubate the plate at 30 &#176;C O/N. This ensures that the mutant is well isolated. Divide the plate into thirds and streak out each colony in one section.</li>
	<li>Spot colonies from the streaked out colonies derived from step 3.12 onto a fresh LB-kan plate to form a grid as in steps 2.1-2.4. Each mutant is being re-tested in triplicate.</li>
	<li>Replicate plate again as in steps 3.1-3.10 to confirm the auxotroph phenotype.</li>
</ol>
4. Gene Rescue
<ol>
	<li>Grow a 3 ml O/N culture of an isolated mutant colony from step 3.13 in liquid LB-kan medium at 30 &#176;C. Purify genomic DNA from 1 ml of culture using a commercially available genomic DNA purification kit.</li>
	<li>Set up a mixture of 0.025 U BfuC I restriction endonuclease and 14 &#181;l of 1 &#956;g/&#956;l genomic DNA in a 20 &#181;l reaction on ice. Since BfuC I cuts the transposon at twelve different sites, it may be more efficient to use restriction endonuclease, Bfa I or Hae III, which cuts the transposon at two and three different sites, respectively.</li>
	<li>Incubate the reaction at 37 &#176;C for 25 min and then at 80 &#176;C for 20 min to inactivate the enzyme. Analyze 3 &#181;l of the partially digested DNA on a 0.8% agarose gel (Figure 3). A successful partial digestion results in a smear from above 10 kb down to the bottom of the gel.</li>
	<li>Ligate 15 &#181;l of partially digested genomic DNA using 800 units of T4 DNA ligase in a 500 &#181;l reaction. Incubate the reaction O/N at 4 &#176;C. The large reaction volume maximizes the ligation of single fragments to themselves and minimizes interactions between two or more DNA fragments.</li>
	<li>Precipitate the ligated DNA by adding 50 &#181;l of 3 M sodium acetate, pH 5.5, and 1 ml of 95% ethanol and incubating it at -20&#176;C for 20 min.</li>
	<li>Microfuge the DNA at 4 &#176;C and 13,500 x g for 10 min, wash the pellet with 70% ethanol, dry it in a centrifugal vacuum concentrator, and resuspend it in 10 &#181;l of nuclease free water. Alternatively, the DNA may be air dried at RT for 15 min.</li>
	<li>Use 4 &#181;l of resuspended DNA to transform E. coli strain ECD100D pir or ECD100D pir116 by electroporation as in section 1. The R6k&#947; replication origin requires a bacterial strain with a pir gene to replicate6. The pir strain results in low copy plasmids, and the pir116 strain contains a pir mutant gene that results in high copy plasmids. Each transformant contains a new plasmid with the transposon and a region of the interrupted chromosome (Figure 1E).</li>
	<li>Inoculate 5 ml of LB-kan medium with a single colony, grow it O/N with shaking at 120 rpm and 37 &#176;C, and purify plasmid DNA from the entire O/N culture using a commercial kit.</li>
	<li>Digest 14 &#181;l of the plasmid using the restriction endonuclease Xho I. Make sure the final volume of the reaction is 20 &#181;l. This enzyme recognizes a site in the middle of the transposon at the 5&#8217; end of the kanamycin resistance gene. Analyze 10 &#181;l of the undigested and digested plasmid on a 0.8% agarose gel (Figure 3). The plasmid consists of the 2 kb transposon plus flanking host DNA.</li>
</ol>
5. DNA Sequencing
<ol>
	<li>Sequence the plasmid using a commercially available sequencing kit, primers that are homologous to each end of the transposon, and a capillary sequencing analysis system. Alternatively, the plasmid may be shipped to an outside institution for sequencing.</li>
	<li>Use the molecular weight standard (1 kb ladder) in the gel to estimate the size of the plasmid. Then, estimate the number of nanograms (ng) of plasmid that is required for 50 fmol.</li>
	<li>Measure the concentration of the plasmid DNA using a spectrophotometer at wavelengths of 260 nm (A260) and 280 nm (A280). Ensure the light path length through the cuvette is 1 cm. Determine the concentration by multiplying the absorbance at 260 nm by 50 ng/&#181;l. High quality plasmid DNA will have an A260/ A280 ratio between 1.8 and 2.0.</li>
	<li>The volume of DNA required for each sequencing reaction is the number of ng required for 50 fmol divided by the concentration of the plasmid. Mix the volume of required plasmid with water to bring the total volume up to 10 &#181;l.</li>
	<li>Heat the plasmid DNA/water mixture at 96 &#176;C for 1 min and then cool it to RT.</li>
	<li>Add 8 &#181;l of master mix from the sequencing kit and 2 &#181;l of 1.6 &#956;M of one of the sequencing primers.</li>
	<li>Follow the protocol from the sequencing kit for the thermal cycler program and reaction cleanup.</li>
	<li>Analyze each sample using a capillary DNA analysis system.</li>
	<li>Use a commercially available software package to view the DNA sequence. Search for the sequence, &#8220;5&#8217;-GAGACAG-3&#8217;,&#8221; which is the last 7 bp of the transposon (Figure 4). The sequence before the 5&#8217; end of this 7 bp segment belongs to the transposon. The sequence after the 3&#8217; end of this 7 bp segment belongs to the interrupted gene.</li>
	<li>Determine the possible function of the interrupted gene using the Basic Local Alignment Search Tool (BLAST) 7,8. Use the following link: <a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&amp;BLAST_PROGRAMS=megaBlast&amp;PAGE_TYPE=BlastSearch" target="_b;ank">http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&#38;BLAST_PROGRAMS=megaBlast&#38;PAGE_TYPE=BlastSearch</a>. Paste the sequence of the interrupted gene into the query box, select &#8220;others&#8221; under the database and click on &#8220;BLAST&#8221; at the bottom of the page. When the result appears, scroll down the page to view the alignment results (Figure 5).</li>
</ol>

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The author has nothing to disclose.

Transposon mutagenesis using a transposome is an efficient tool for generating auxotrophs in Enterobacter sp. YSU and other types of Gram negative and Gram positive bacteria3,4. The process was initiated by introducing the Tn5-derived transposome into the host by electroporation. To identify colonies with inserts, the untransformed target strain had to be sensitive to kanamycin in order to select for the resistance marker carried by the transposon. Some hosts produce restriction endonucleases...

Prototrophic bacteria grow in M-9 minimal salts medium containing glucose (M-9 medium), converting glucose through central carbon metabolism pathways to generate precursors, such as amino acids, nucleic acids and vitamins, for biosynthesis1. M-9 medium contains ammonium chloride as a nitrogen source, sodium and potassium phosphate as a buffer and phosphorous source, magnesium sulfate as a sulfur source and glucose as a carbon and energy source. Luria-Bertani (LB) medium is rich in amino acids from tryptone and in vitamins and growth factors from yeast extract. It supports the growth of auxotrophs that cannot synthesize amino acids, vitamins and other growth factors required for growth on M-9 medium. Thus, prototrophs will grow in LB and M-9 medium, whereas auxotrophs will grow in LB medium but not in M-9 medium. By introducing mutations into a prototrophic population of bacteria and identifying mutated genes that cause auxotrophic phenotypes, it is possible to obtain a better understanding of the metabolism in a bacterial strain.
Transposon mutagenesis can be used to identify many of the genes required for growth on glucose in M-9 medium. Transposons insert themselves randomly into the host genome2. By spotting transposon transformants on LB agar plates and replica plating them onto M-9 medium agar plates, it is possible to screen for auxotrophs. Interrupted genes are identified through gene rescue. This study uses a commercially available Tn5-derived transposome which is shipped as a solution of a linear transposon DNA segment mixed with transposase protein. The DNA segment lacks a transposase gene but contains a gene for kanamycin resistance, an R6K&#947; replication origin and two mosaic motifs which are DNA sequences for transposase binding at each end of the segment3,4. Since the transposase protein is added directly to the DNA, the DNA segment alone is defined as the transposon, and the DNA/transposase protein complex is defined as the transposome. The transposome is transformed by electroporation5 into a kanamycin sensitive host (Figure 1A). Colonies that grow on LB agar plates containing kanamycin (LB-kan) have transposon inserts (Figure 1B), and replica plated transformants that fail to grow on M-9 medium agar plates containing kanamycin (M-9-kan) are auxotrophs (Figire 1C). Genomic DNA from a mutant is purified and partially digested with the 4-base cutting restriction endonuclease, BfuC I (Figure 1D). The ligated DNA is transformed into a strain of Escherichia coli (E. coli) that contains the pir gene (Figure 1E). This gene allows the new plasmid containing the transposon and flanking host chromosomal region to replicate in E. coli6. The kanamycin resistance gene serves as a selectable marker for the new plasmid. Finally, sequencing using primers complementary to each end of the transposon and Basic Local Alignment Search Tool (BLAST) analysis7,8 of the resulting sequence are used to determine the identity of the interrupted genes.
This transposome mutagenesis strategy provides three advantages3. First, since the transposase protein is bound directly to the transposon, insertion does not depend on expression of the transposase gene within the host. Once the transposon incorporates itself into the host genome, the transposase is degraded, preventing additional movement of the transposon. However, additional movement cannot be prevented if the host possesses an endogenous Tn5 transpositional element. Second, introduction of the transposome by electroporation makes it possible to use it in a wide variety of hosts. It also eliminates the need to introduce the transposon by bacterial conjugation or by viral infection. Both processes require host susceptibility. Third, inclusion of the kanamycin resistance gene and the R6K&#947; replication origin in the transposome makes it easier to identify the interrupted gene. Transposon-interrupted regions can be stored and sequenced as plasmids, eliminating the need to use the inverse polymerase chain reaction (PCR) technique for gene identification.
The protocol presented in this video describes each step for transposon mutagenesis of Enterobacter sp. YSU9 using a transposome from its introduction into the bacterial cells to the identification of the putative gene it interrupted. In addition to previously published protocols3,4,10, detailed methods for using replica plating to screen for auxotrophs are presented. This mutagenesis technique may be used for investigating other phenotypes, such as antibiotic and metal resistances, in different types of bacteria, for identifying the minimal number of genes required for growth under defined culture conditions in synthetic biology studies, or for teaching a laboratory component of a genetics or microbial physiology course.&#160;&#160;

<table><tbody><tr><td>EZ-Tn5 R6K&amp;gamma;ori/KAN-2 Tnp Transposome Kit</td><td>Epicentre (Illumina)</td><td>TSM08KR</td><td></td></tr><tr><td>EC100D pir&lt;sup&gt;+&lt;/sup&gt; Electrocompetent E. coli</td><td>Epicentre (Illumina)</td><td>ECP09500</td><td>Capable of replicating plasmids with an R6K&amp;gamma; replication origin at a low copy number</td></tr><tr><td>EC100D pir-116 Electrocompetent E. coli</td><td>Epicentre (Illumina)</td><td>EC6P095H</td><td>Capable of replicating plasmids with an R6K&amp;gamma; replication origin at a high copy number</td></tr><tr><td>5X M-9 Salts</td><td>Thermo Fisher</td><td>DF048517</td><td></td></tr><tr><td>Lennox LB Broth</td><td>Thermo Fisher</td><td>BP1427-2</td><td></td></tr><tr><td>Agar</td><td>Amresco, Inc.</td><td>J637-1KG</td><td>Solid media contained 1.6% (w/v) agar</td></tr><tr><td>Kanamycin Sulfate&amp;nbsp;</td><td>Amresco, Inc.</td><td>0408-25G</td><td>When required, media contained 50 &amp;mu;g/ml kanamycin sulfate</td></tr><tr><td>D-Glucose Monohydrate</td><td>Amresco, Inc.</td><td>0643-1KG</td><td></td></tr><tr><td>Yeast Extract</td><td>Amresco, Inc.</td><td>J850-500G</td><td></td></tr><tr><td>Tryptone</td><td>Amresco, Inc.</td><td>J859-500G</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P4504-500G</td><td></td></tr><tr><td>NaCl</td><td>Amresco, Inc.</td><td>X190-1KG</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>BP214-500</td><td></td></tr><tr><td>MgSO&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>BP213-1</td><td></td></tr><tr><td>Super Optimal Broth with Catabolite Repression (SOC) medium</td><td></td><td></td><td>0.5% (w/v) yeast extract, 2% (w/v) tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 20 mM MgSO&lt;sub&gt;4&lt;/sub&gt; and 20 mM Glucose</td></tr><tr><td>BfuC I</td><td>NEB</td><td>R0636S</td><td>Partial digestion of genomic DNA</td></tr><tr><td>Xho I</td><td>NEB</td><td>R0146S</td><td>Plasmid digestion</td></tr><tr><td>T4 DNA Ligase</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>Nuclease Free Water</td><td>Amresco, Inc.</td><td>E476-1L</td><td>For dissolving precipitated DNA and restriction endonuclease reactions</td></tr><tr><td>GenomeLab DTCS - Quick Start Kit</td><td>Beckman Coulter</td><td>608120</td><td>DNA sequencing</td></tr><tr><td>Wizard Genomic DNA Purification Kit</td><td>Promega</td><td>A1120A</td><td></td></tr><tr><td>Wizard Plus SV Minipreps DNA Purification System</td><td>Promega</td><td>A1460</td><td>Plasmid purification</td></tr><tr><td>Replica Plating Block</td><td>Thermo Fisher</td><td>09-718-1</td><td></td></tr><tr><td>Velveteen Squares</td><td>Thermo Fisher</td><td>09-718-2</td><td>Replica plating</td></tr><tr><td>PetriStickers</td><td>Diversified Biotech</td><td>PSTK-1100</td><td>Grid for replica plating</td></tr><tr><td>Petri Dishes</td><td>Thermo Fisher</td><td>FB0875712</td><td></td></tr><tr><td>Gene Pulser II</td><td>BioRad</td><td></td><td>Electroporation</td></tr><tr><td>Electroporation Cuvettes &amp;ndash; 2 mm</td><td>BioExpress</td><td>E-5010-2</td><td></td></tr><tr><td>CentriVap DNA Vacuum Concentrator</td><td>Labconco</td><td>7970010</td><td>Drying DNA</td></tr><tr><td>CEQ 2000XL DNA Analysis System</td><td>Beckman Coulter</td><td></td><td>DNA sequencing</td></tr><tr><td>Vector NTI Advance 11.5.0</td><td>Life Technologies</td><td>12605099</td><td>DNA sequence analysis</td></tr></tbody></table>

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.

Transformation of Enterobacter sp. YSU by electroporation with the transposome initiated random genome insertion into the host genome (Figure 1A,B). A successful electroporation yielded several thousand transformants which grew on LB-kan plates. To obtain 300-400, well-spaced colonies per plate, the amount of transformation mixture spread on each LB-kan agar plate was optimized. Each transformant contained at least one transposon insert, but it was not clear if an important gene for growth on M-...

Watch this Scientific Journal Video about Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis at JoVE.com

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.

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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.8K Views.  Youngstown State University. 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.

Video: Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

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 ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Immunology and Infection

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.

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.

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 ...

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 ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Bioengineering

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Summary

Explore More Videos

Chapters in this video

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

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

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

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

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

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions