Team:Calgary/Project/FRED/Detecting

From 2012.igem.org

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TITLE=A Transposon Screen for Detecting Naphthenic Acids|
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TITLE=A Transposon-Mediated Mutant Library for Toxin Detection|
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<img src="http://2012.igem.org/wiki/images/5/52/UCalgary2012_FRED_Detecting.png" style="float: right; padding: 10px; height: 280px;"></img>
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This year, our team wanted to identify a novel responsive element capable of detecting and quantifying naphthenic acids (NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing NAs, the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of naphthenic acids.
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This year, our team wanted to identify a novel responsive element capable of detecting and quantifying different tailings ponds toxins (e.g. naphthenic acids, NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing a variety of toxic compounds (e.g. NAs), the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of these toxins.
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<h2>Transposons: What, How, Why?</h2>
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The transposable element (TE), Tn5, is a conservative transposon that can insert a segment of genes bordered by specific 19bp insertion sequences from one part of the genome (e.g. plasmid vector) randomly to another location like a chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by <i>tnp</i> gene included in the TE. Using the appropriate selective pressure, the insertion can be maintained permanently in the genome.</p>
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</html>[[File:Transposon.jpg|thumb|700px|center|Figure 1: "Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome. Modified from Transposons: Shifting Segments of the Genome" by McGraw Hill]]<html>
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<br>
 
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<h3>Transposons: What, How, Why?</h3>
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<p align="justify">By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Using a <a href="http://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">bipartite-mating (conjugation) method</a> to transfer the TE vector into the organism of choice is an efficient method for creating the massive number of mutants required.</p>
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The transposable element (TE), Tn5, is a conservative transposon that is able to insert a segment of genes bordered by specific 19bp insertion sequences (IS) from one part of the genome (e.g. plasmid vector) randomly to another location, such as the chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by Tnp gene included in the TE. </p><p align="justify">By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Another advantage of using a transposon approach is that it creates a saturating library of mutants where all possible genetic elements responding to a certain environmental stimuli can be identified. Therefore, saturated genome-wide mutant libraries generated by transposon-mediated mutagenesis are powerful tools that serve our purpose in identifying unknown non-essential genes (e.g. metabolism of alternative carbon sources) and characterizing the function of these unknown genes that are involved in NA detection and degradation. However, due to the random nature of TE insertions, two considerations need to be taken. First, to screen the entire genome of an organism, a large number of mutants needs to be generated, which is time-consuming. Second, the TE insertion is not permanent, and thus, the TE may move to another location after the first insertion. The first concern can ameliorated by using a bipartite-mating (conjugation) method to transfer the TE vector into the organism of choice, which is efficient at creating a massive library of mutants (see the method design section for details). The second concern is addressed by maintaining mutants under the appropriate selective pressure, ensuring the reporter gene is still fused to a promoter element upstream by screening for the reporter gene products (e.g. lacZ producing an insoluble blue pigment in the presence of X-Gal), and isolating the mutants' genomic DNA in a timely fashion.
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Due to the complexity of biological systems, our team focused our efforts on utilizing a system for identification of promoter elements that respond specifically in the presence of environmental stimuli. Our hypothesis requires that the organisms we use respond specifically to particular toxins and result in upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules may play a role in toxin sensing, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the degradation mechanism by elucidating the genes involved.
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Due to the complexity of biological systems, our team focused our efforts on identifying a system for identification of promoter elements that respond in the presence of environmental stimuli. Identifying such a system for naphthenic acids, if one exists, would allow for a specific response to be generated to many different kinds of naphthenic acids. Our hypothesis requires that the organisms we use respond specifically to naphthenic acids and result in specific upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules within a NA-degrading organism may play role in the sensing and modification of NAs, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the mechanism of NA degradation by elucidating the genes involved. If this can be accomplished, then we can easily construct an organism capable of specifically and robustly detecting a variety of NAs, and modify them to reduce their toxicity. The transposon system not only allow the identification of such as sytstem, but also allow the resultant system to be broad enough to detect a variety of NAs, instead of simply model compounds.
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<h3>Naphthenic Acid Degrading Organism Used</h3>
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<h2>Toxin-Degrading Organism Used</h2>
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Pseudomonads are species of aerobic bacteria that have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs (Ramos-Padrón et al. 2010; Herman et al., 1994; Del Rio et al., 2006; Gieg & Whitby, unpublished, 2012). Since they can degrade NAs, this suggests that there exist a system that actively recognizes and is up-regulated specifically in response to NAs. We wanted to use a commercially available strain of Pseudomonas fluorescens characterized for a response to model NAs (model single- and double-ringed compounds) and NAs isolated from tailings pond water (TPW). The <i>P. fluorescens pf-5</i> strain is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, sequencing data is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occured, and what genes (if present) are located downstream of it.
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<i>Pseudomonas spp. </i>have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs and nitrogen- and sulfur-containing heterocyclic aromatic compounds (Ramos-Padrón <i>et al</i>. 2010; Herman <i>et al</i>., 1994; Del Rio <i>et al</i>., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins.</p><p> We wanted to use a commercially available strain of <i>Pseudomonas fluorescens</i> characterized for a response to toxins found in tailings pond water (TPW). The <i>P. fluorescens </i>PF-5 strain (Paulsen <i>et al</i>., 2005) is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, the genome sequence is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occurred, and what genes (if present) are located downstream of it.
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<h3>Method Design</h3>
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<a name="library"></a><h2>Method Design</h2>
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<h3>Mutant Library Generation</h3>
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To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element)  is introduced into commercially purchased E. coli SM10 donor strain.</p>
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To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element)  is introduced into commercially purchased <i>E. coli SM10</i> donor strain.</p>
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</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|700px|center|Figure 1: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an E. coli origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements]]<html>
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</html>[[File:Transposonproject Tn5OT182constructucalgary.png|thumb|750px|center|Figure 2: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an
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<i> E. coli</i> origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements]]<html>
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<p align="justify">The plasmid contains a RP4 mob conjugation region and a p15A origin of replication (ori), and is engineered to only replicate in <i>E. coli</i>. The TE construct is transferred from the <i>E. coli</i> donor strain to the recipient <i>P. fluorescens </i> PF-5 using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in <i>P. fluorescens</i>, and selected by isolating the recipients that have a genomic TE insertion on Pseudomonas Isolation Agar/PIA with tetracycline. If a promoter element is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which can be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound) (Juers <i>et al</i>., 2012). If the fused promoter is activated in response to a stimulus, then the lacZ enzyme will be produced in response. Mutant strains sensitive to the particular toxic stimulus will appear as blue colonies on the selective plate.</p>
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<h3>Mutant Strain Characterization</h3>
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<p align="justify">Mutants generated are characterized for their roles in the response to toxins with dose response experiments, and compared to general stress-inducing agents (e.g. H<font style="text-transform: lowercase;">2</font>O<font style="text-transform: lowercase;">2</font>) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.</p>
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</p>
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<h3>Self-Cloning and Sequencing</h3>
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<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman and Lamont, 1993). The TE used is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from a desirable mutant is isolated, and restriction digested with BglII (a restriction enzyme that does not cut within the TE but numerous times within the genome). The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into <i>E. coli</i>. Plasmids from the transformed cells contain the TE construct with the upstream and downstream flanking sequencing connected by the BglII restriction site. Sequencing primers designed against the 19 bp recognition sequence in the TE to sequence the isolated plasmids.</p>
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<p align="justify">For a detailed protocol, please consult our <a href="http://2012.igem.org/Team:Calgary/Notebook/Protocols/tnscreen">methods section</a>.</p>
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<h2>Results</h2>
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<h3>Detection by Mutant <i>Pseudomonas fluorescens</i> PF-5</h3>
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<p align="justify">After mating experiments and plating on selective media (Pseudomonas isolation agar, with tetracycline and naphthenic acids), 24 responsive (blue) colonies were found. Screens were conducted on these blue colonies found on selective plates comparing a response in LB and LB with 100mg/L naphthenic acids (both with X-Gal). When results were observed it was found that 4 mutant strains are differentially regulated in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. These colonies were further screened to test the specificity of their responses.</p>
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<p align="justify"></html>[[File:Transposon1initialscreenucalgary.PNG|thumb|500px|center|Figure 3: Transposons: Shifting Segments of the Genome: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 &micro;g/ml. Cells were allowed to grow at 30&deg;C for 16hr. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.]]<html></p><p align="justify">
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<p>The plasmid is engineered in a vector containing a RP4 mob conjugation region, and a p15A origin of replication (ori). The IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR construct is transferred from the <i>E. coli</i> donor strain to the recipient <i>P. fluorescens pf-5</i> using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in <i>P. fluorescens</i>, and selected by isolating the recipients on Pseudomonas Isolation Agar/PIA (the antibiotic Irgasan in this medium kills all other bateria, and only allows the survival of <i>Pseudomonas spp.</i>) and the recipient cells that have a genomic TE insertion using tetracyclin (conjugated plasmids cannot replicate in Pseudomonas, and thus the Pseudomonas that survived must have a TE insertion in the genomic DNA). If a promoter elements is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound). If the fused promoter is activated in response to NAs, then lacZ will be produced in response to NAs. Desirable mutants are selected by plating the conjugated bacteria onto an agar plate containing PIA, tetracyclin (at a concentration that wildtype <i>Pseudomonas fluorescens pf-5</i> is sensitive to), a NA mixture (Acros), and X-Gal. Mutants are sensitive to NAs will appear as blue colonies on the plate.
 
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<p align="justify">Promoters identified by the transposon mutagenesis strategy are characterized for their roles in the response to NA exposure with dose response curves, and compared to general stress-inducing agents (e.g. H2O2) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.</p>
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Screens involving the use of different toxins at environmentally relevant concentrations were performed to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used as one testing condition to determine if the response is simply stress-induced.
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<p align="justify"></html>[[File:Tn5 screen 2nd round colony170.PNG|thumb|600px|center|Figure 4: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p>
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<p align="justify">Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion. IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from the desirable mutant is isolated, and restriction digested with either BglII or XbaI, which are restriction enzymes that do not cut within the transposon but numerous times with the genome. The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into <i>E. coli Top10</i>, and plated on LB agar with tetracyclin. The transformed cells now have plasmids containing the TE construct with the upstream and downstream flanking sequencing connected by the restriction site (of BglII or XbaI). Plasmids isolated from the transformed cells are used for sequencing. Sequencing primers are designed against the 19 bp recognition sequence in the TE to sequence outward from the transposon.</p>
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<p align="justify"></html>[[File:170-1data.png|thumb|650px|center|Figure 5: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p>
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<p align="justify">For a detailed protocol, please consult our methods section.</p>
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<p align="justify"></html>[[File:Tn5 screen 2nd screen Colony66.PNG|thumb|600px|center|Figure 6: Second Screen- 66-1.  Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p>
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<h2>Results</h2>
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<p align="justify"></html>[[File:66-1 1-100 data.png|thumb|650px|center|Figure 7: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.]]<html></p>
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<h3>Detection by Mutant <i>Pseudomonas fluorescens</i> PF-5</h3>
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<p align="justify">
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<p></html>[[File:|thumb|700px|center|Figure: Initial Hit Screen Comparison Pictures (24 colony)]]<html></p>
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From these screens, it was seen that both colony 66-1 and colony 170-1 appear to respond to toxins when compared to a response in LB media. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The results of this can be seen below.</p>
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<p></html>[[File:|thumb|700px|center|Figure: Second Screen- 66 series with pic]]<html></p>
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<p align="justify">
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<p></html>[[File:|thumb|700px|center|Figure: Second Screen- 66 series data]]<html></p>
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</html>[[File:Ucalgary2012-FreddetectingTRANSPOSONstresstest.png|thumb|800px|center|Figure 8: Stress response screen on <i>P. fluorescens</i> Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions (shown are '''A:''' 66-1 undiluted, '''B:''' 66-1 at 1/10 dilution, '''C:''' 170-1 undiluted, '''D:''' 170-1 at 1/10 dilution)  into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 uL of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.]]<html><p>
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<p></html>[[File:|thumb|700px|center|Figure: Second Screen- 170 series with pic]]<html></p>
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<p></html>[[File:|thumb|700px|center|Figure: Second Screen- 170 series data]]<html></p>
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These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.</p>
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<h2>Promoter Constructs Isolated</h2>
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<p align="justify">To determine the location of the transposon insertion, we utilized the self-cloning properties of the transposon. By digesting the genome, religating, and transforming the ligated genomic fragments into <i>E. coli</i>, plasmids containing the transposon and flanking gene sequences were isolated. These plasmids have been isolated and sent for sequencing. However, we are having difficulty with getting sequencing reactions to produce a read. The results so far are a promising step towards finding a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds. </p><p>
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Our next steps were to test these strains in conjunction with our electrochemical detector as well as see if they could detect tailings toxins. The results of this can be seen on the <a href="http://2012.igem.org/Team:Calgary/Project/Synergy">Synergy page</a>.</p>
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<h3>Promoter Constructs Isolated</h3>
 
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<h3>References</h3>
 
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<p>
 
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(1) Del Rio LF, Hadwin AK, Pinto LJ, MacKinnon MD, Moore MM. Degradation of naphthenic acids by sediment micro-organisms. J Appl Microbiol 2006 Nov;101(5):1049-1061. </p>
 
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<p>
 
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(2) Herman DC, Fedorak PM, MacKinnon MD, Costerton JW. Biodegradation of naphthenic acids by microbial populations indigenous to oil sands tailings. Can J Microbiol 1994 Jun;40(6):467-477. </p>
 
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<p>
 
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(3) Holowenko FM, Mackinnon MD, Fedorak PM. Naphthenic acids and surrogate naphthenic acids in methanogenic microcosms. Water Res 2001 Aug;35(11):2595-2606. </p>
 
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<p>
 
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(4) Lewenza S, Falsafi RK, Winsor G, Gooderham WJ, McPhee JB, Brinkman FS, et al. Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes. Genome Res 2005 Apr;15(4):583-589. </p>
 
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<p>(5) Ramos-Padron E, Bordenave S, Lin S, Bhaskar IM, Dong X, Sensen CW, et al. Carbon and sulfur cycling by microbial communities in a gypsum-treated oil sands tailings pond. Environ Sci Technol 2011 Jan 15;45(2):439-446.
 
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(6) Reznikoff WS. Transposon Tn5. Annu Rev Genet 2008;42:269-286. </p>
 
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<p>
 
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(7) Sheridan DL, Hughes TE. A faster way to make GFP-based biosensors: two new transposons for creating multicolored libraries of fluorescent fusion proteins. BMC Biotechnol 2004 Aug 18;4:17. </p>
 
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<p>
 
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(8) So J. Mini-transposon Tn5gfp constructs for differential tagging of microorganisms. Biotechnology and Bioprocess Engineering 1999;4(2):154-156. </p>
 
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<p>
 
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(9) Timms-Wilson TM, Bailey MJ. Reliable use of green fluorescent protein in fluorescent pseudomonads. J Microbiol Methods 2001 Jul 30;46(1):77-80. </p>
 
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<p>(10) Zhang X, Wiseman S, Yu H, Liu H, Giesy JP, Hecker M. Assessing the toxicity of naphthenic acids using a microbial genome wide live cell reporter array system. Environ Sci Technol 2011 Mar 1;45(5):1984-1991.
 
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Latest revision as of 03:10, 27 October 2012

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A Transposon-Mediated Mutant Library for Toxin Detection

This year, our team wanted to identify a novel responsive element capable of detecting and quantifying different tailings ponds toxins (e.g. naphthenic acids, NAs) in solution. While numerous studies have begun to identify species of bacteria capable of surviving and sensing a variety of toxic compounds (e.g. NAs), the degradation pathways have not yet been fully characterized. Therefore, we needed to design and implement novel approaches to efficiently isolate the genetic elements that detect and potentially lead to the breakdown of these toxins.

Transposons: What, How, Why?

The transposable element (TE), Tn5, is a conservative transposon that can insert a segment of genes bordered by specific 19bp insertion sequences from one part of the genome (e.g. plasmid vector) randomly to another location like a chromosome (Reznikoff, 2008). The transposition event is catalyzed by a transposase enzyme encoded by tnp gene included in the TE. Using the appropriate selective pressure, the insertion can be maintained permanently in the genome.

Figure 1: "Transposition reaction from plasmid entry into the recipient cell to integration of the transposon into the genome. Modified from Transposons: Shifting Segments of the Genome" by McGraw Hill

By inserting a vector construct containing the TE with selectable markers (such as tetracyclin resistance and lacZ) into an organism with a desirable phenotype, we can find out what genetic elements (e.g. genes and promoters) are responsible for that particular function. This can happen via a random insertion of a TE containing a promoterless reporter gene downstream of promoter elements that creates a transcriptional fusion, providing activity in response to specific environmental stimuli. Using a bipartite-mating (conjugation) method to transfer the TE vector into the organism of choice is an efficient method for creating the massive number of mutants required.

Due to the complexity of biological systems, our team focused our efforts on utilizing a system for identification of promoter elements that respond specifically in the presence of environmental stimuli. Our hypothesis requires that the organisms we use respond specifically to particular toxins and result in upregulation of metabolic genes with little background effect in the cell. We recognize that any number of biological molecules may play a role in toxin sensing, such as enzymes, transcription factors, and even RNA elements (e.g. riboswitches). However, the identification of a promoter sequence takes us further in that we can better understand the degradation mechanism by elucidating the genes involved.


Toxin-Degrading Organism Used

Pseudomonas spp. have been isolated from oil sands tailings ponds and shown to biodegrade model and tailings-associated NAs and nitrogen- and sulfur-containing heterocyclic aromatic compounds (Ramos-Padrón et al. 2010; Herman et al., 1994; Del Rio et al., 2006; Gieg & Whitby, unpublished, 2012). This suggests that there exists systems that detect and up-regulate transcription specifically in response to these toxins.

We wanted to use a commercially available strain of Pseudomonas fluorescens characterized for a response to toxins found in tailings pond water (TPW). The P. fluorescens PF-5 strain (Paulsen et al., 2005) is reported to survive in and degrade a commercial mixture of naphthenic acids (Acros) (Gieg & Whitby unpublished, 2012). Moreover, the genome sequence is available for this strain with annotations (Pseudomonas Genome Database V2, http://pseudomonas.com/). This allows us to use sequencing data from the mutants and identify where in the genome the TE insertion occurred, and what genes (if present) are located downstream of it.


Method Design

Mutant Library Generation

To construct the promoter library, a pOT182 vector construct (containing a IR-lacZ-Amp-pMB1ori-TetA-TetR-Tnp-IR transposable element) is introduced into commercially purchased E. coli SM10 donor strain.

Figure 2: The transposable Tn5 element used in the pOT182 plasmid, containing a lacZ reporter gene, ampicillin and tetracycline resistance, an E. coli origin of replication for use during downstream sequencing protocols, and transposase. The genes are flanked by the transposon insertion elements

The plasmid contains a RP4 mob conjugation region and a p15A origin of replication (ori), and is engineered to only replicate in E. coli. The TE construct is transferred from the E. coli donor strain to the recipient P. fluorescens PF-5 using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in P. fluorescens, and selected by isolating the recipients that have a genomic TE insertion on Pseudomonas Isolation Agar/PIA with tetracycline. If a promoter element is fused upstream of the TE construct, then promoter activation will turn on the expression of lacZ, which can be detected by the degradation of a colorless compound, X-Gal, to an insoluble blue pigment product (an indoxyl compound) (Juers et al., 2012). If the fused promoter is activated in response to a stimulus, then the lacZ enzyme will be produced in response. Mutant strains sensitive to the particular toxic stimulus will appear as blue colonies on the selective plate.

Mutant Strain Characterization

Mutants generated are characterized for their roles in the response to toxins with dose response experiments, and compared to general stress-inducing agents (e.g. H2O2) and compounds such as fatty acids to ensure the specificity of the response. These measurements help to determine thresholds of detection, robustness of the signal, and specificity of response. The dose response curves will also assess the usefulness of correlating the concentration of NA to the level of response, and the possibility of measuring NA concentrations in a sample, rather than simply by presence/absence.

Self-Cloning and Sequencing

Last, self-cloning techniques are used to identify the upstream and downstream sequences from the TE insertion (Merriman and Lamont, 1993). The TE used is a self-cloning construct because it contains all the elements required for plasmid replication (i.e. origin of replication) and selection (Tet resistance). Genomic DNA from a desirable mutant is isolated, and restriction digested with BglII (a restriction enzyme that does not cut within the TE but numerous times within the genome). The resulting fragments may contain the TE construct with flanking sequences. The genomic fragments are circularized by self-ligation and transformed into E. coli. Plasmids from the transformed cells contain the TE construct with the upstream and downstream flanking sequencing connected by the BglII restriction site. Sequencing primers designed against the 19 bp recognition sequence in the TE to sequence the isolated plasmids.

For a detailed protocol, please consult our methods section.

Results

Detection by Mutant Pseudomonas fluorescens PF-5

After mating experiments and plating on selective media (Pseudomonas isolation agar, with tetracycline and naphthenic acids), 24 responsive (blue) colonies were found. Screens were conducted on these blue colonies found on selective plates comparing a response in LB and LB with 100mg/L naphthenic acids (both with X-Gal). When results were observed it was found that 4 mutant strains are differentially regulated in response to naphthenic acids: 66-1, 66-2, 170-1, and 199-1. These colonies were further screened to test the specificity of their responses.

Figure 3: Transposons: Shifting Segments of the Genome: Initial Hit Screen Comparison Pictures. Colonies were inoculated in duplicate into both LB media, and LB media containing 100 mg/L ACROS commercial naphthenic acids. X-gal was added to the media at a final concentration of 200 µg/ml. Cells were allowed to grow at 30°C for 16hr. Blue coloration indicates levels of LacZ production. 4 colonies (66-1, 66-2, 170-1, and 190-1) showed differential regulation in naphthenic acids.

Screens involving the use of different toxins at environmentally relevant concentrations were performed to determine if the sensing response was specific to naphthenic acids, or if a sensory response to general toxins had been found. In addition, hydrogen peroxide was used as one testing condition to determine if the response is simply stress-induced.

Figure 4: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 12h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.

Figure 5: Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.

Figure 6: Second Screen- 66-1. Second Screen- 170-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. After 24h, deeper blue coloration was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.

Figure 7: Second Screen- 66-1. Cells were inoculated in duplicate at different dilutions into LB as a control, and LB containing different toxin compounds at environmental concentrations. Hydrogen peroxide was used to rule out a stress response. X-gal was added to the media. Absorbance was read at 615nm (maximal absorbance of X-gal) every hour. Higher absorbance was observed in the toxin wells compared to the LB control. The cells did not grow in the hydrogen peroxide due to an excessively high concentration.

From these screens, it was seen that both colony 66-1 and colony 170-1 appear to respond to toxins when compared to a response in LB media. In order to test the specificity of this response, an additional screen was performed using varying concentrations of hydrogen peroxide (to rule out activation by a general stress response in the cell) in addition to decanoic acid at a comparable concentration to that of the naphthenic acids used (to rule out activation due to sensing fatty acid compounds). The results of this can be seen below.

Figure 8: Stress response screen on P. fluorescens Pf5 transposon mutants. Cells were inoculated in duplicate at different dilutions (shown are A: 66-1 undiluted, B: 66-1 at 1/10 dilution, C: 170-1 undiluted, D: 170-1 at 1/10 dilution) into LB as a control, LB containing varying concentrations of hydrogen peroxide, LB containing naphthenic acids at an environmental concentration, and LB containing decanoic acid at the same concentration as the naphthenic acids. 2 uL of 20mg/ml X-gal was added to the media and absorbance was read at 615nm (maximal absorbance of X-gal) every 4 hours for 12h. Higher absorbance was observed in the NA wells compared to the LB control, hydrogen peroxide, and decanoic acid for colony 66-1. Colony 170-1 showed a repressed response to naphthenic acids when compared to the LB control.

These results show that colony 66-1 gives a response to naphthenic acids and other toxins that is not simply a response to fatty acids or a general stress response. Unfortunately, colony 170-1 does not show a useful reporter response.

Promoter Constructs Isolated

To determine the location of the transposon insertion, we utilized the self-cloning properties of the transposon. By digesting the genome, religating, and transforming the ligated genomic fragments into E. coli, plasmids containing the transposon and flanking gene sequences were isolated. These plasmids have been isolated and sent for sequencing. However, we are having difficulty with getting sequencing reactions to produce a read. The results so far are a promising step towards finding a sensory element for our reporter system that would allow for the detection of various toxins in tailings ponds.

Our next steps were to test these strains in conjunction with our electrochemical detector as well as see if they could detect tailings toxins. The results of this can be seen on the Synergy page.