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A Transposon Screen for Detecting Naphthenic Acids

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.

Transposons: What, How, Why?

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).

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. 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.

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 elucidation the mechanism of NA degradation and 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.

Naphthenic Acid Degrading Organism Used

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 P. fluorescens pf-5 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, 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.

Method Design

To construct the promoter library, a mini-transposon 5 (Tn5)-GFP-RFP-KmR (kanamycin resistance) construct is introduced into commercially purchased E. coli SM10 donor strain (So, 1999; Tang et al., 1999). The transposable element (TE), Tn5, is a conservative transposon that is able to insert a segment of genes bordered by specific 19bp recognition sequences (RS) from one part of the genome (e.g. plasmid vector) randomly to another location (Reznikoff, 2008). The construct is engineered in a pir+ vector containing a RP4 mob conjugation region, and R6K origin of replication (ori) (So 1999). The pir gene ensures that plasmids with R6Kori regions can only replicate in E. coli (So, 1999). The Tn5-GFP-RFP-KmR construct is transferred from the E. coli donor strain to the recipient P. fluorescens PF5 *above you said a different strain? strain using bipartite mating via conjugation (enabled by the RP4 mob region). A random genomic library of transposon insertions is created in P. fluorescens, and screened by selecting fluorescent colonies that can grow on kanamycin containing agar plates under UV light and placing them in 96-well plates. Using a plate-reader, the fluorescence of the conjugant is measured, and those with the strongest responses are further characterized and sequenced. Sequence primers are designed against the 19 bp recognition sequence surrounding the transposed genes (i.e. GFP-RFP-KmR) (So, 1999). The construct (along with any upstream promoter regions) are isolated and circularized before sequencing. Foreseen challenges include the necessity to screen large number of clones and the background fluorescence in P. fluorescens. However, the use of the GFP/RFP system that is amenable to high throughput screening via plate readers will mitigate that difficulty. Also, as P. fluorescens produces a green fluorescent pigment, the use of RFP and other fluorescent proteins while setting specific filters on the plate reader will circumvent the problem (Sheridan & Hughs, 2004). Also, the addition of iron supplement to the growth media has been shown to reduce the endogenous fluorescence, allowing GFP fluorescence to be read accurately (david sent me a paper on this, we might want to use this approach. I attatched it to the email)

Promoters identified by the transposon mutagenesis strategy will be characterized for their roles in the response to NA exposure with dose response curves, and compared to general stress-inducing agents (e.g. H2O2). These measurements will help to determine thresholds of detection, robustness of 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.