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| Tracy Neher (Ph.D. Candidate in Biochemistry and Molecular Biology.)
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The immediate objective of the research Tracy is performing is to determine the mechanism of assimilation of naphthalene, a polycyclic aromatic hydrocarbon (PAH), by cells of Pseudomonas fluorescens . Our laboratory has previously proposed that naphthalene is actively transported and, therefore, it should be possible to identify a transport protein specific for the transport of naphthalene. There are many transport proteins present in cells, not all involved in the uptake of PAH's. Therefore, as much background as possible must be eliminated before attempting to isolate the transport protein. To eliminate this background, Tracy has isolated the mRNA populations, from the total RNA populations, of control (glucose grown) and experimental (naphthalene grown) cells. RT-PCR (Reverse transcriptase coupled with PCR) is used to obtain the cDNA's from the mRNA of each cell population and suppressive, subtractive hybridization is then performed to obtain only the differentially |
| expressed naphthalene genes. The next step is to develop a cDNA library of the differentially expressed genes by ligating them into the cloning vector pGEM-T. Tracy has completed this cDNA library and has completely sequenced the cDNA contained in 122 of the isolated E. coli transformants that she selected. All sequencing has been done using the BigDye termination technique and has been completed on the ABI systems sequencer owned by the department. |
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| Figure 6: Total RNA and mRNA Electrophoresis Results. Notice after isolating the mRNA from
the total RNA the 23S and 16S rRNA bands have disappeared. |
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Figure 7: Suppressive Subtractive Hybridization Schmatic
Shown are the hybridization steps that eliminate any similar sequences present in the control and experimental populations. PCR templates that will be exponentially amplified will result in the differentially expressed naphthalene genes.
(click images to enlarge) |
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Figure 8: pGEM-T Vector Map.
The plasmid comes in a linear form and has an ampicillin resistant gene encoding a B-Lactamase. Once electroporation is performed with pGEN-T and E.coli JM109, color screening is done to identify recombinant clones. White colonies have a cDNA fragment inserted white blue colonies have no insertion. The insertion disrupts the B-glactosidase gene and the colonies appear white. |
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| After completing a series of Blast analyses on the sequences contained in the cDNA library, Tracy selected 5 sequences as potential candidates for transport proteins involved in the assimilation of naphthalene. However, these identifications are only tentative and now the complete sequences of the identified genes must be obtained.
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To obtain these complete gene sequences, Tracy is using the powerful technique of Genome Walking. A schematic of this technique is shown to the left. With this approach, the organism's genomic DNA is treated separately with four different restriction enzymes and the restriction fragments generated by this treatment are then ligated to special adaptors (AP1, etc.) that have known primer sequences. Thus, you have produced what is referred to as a genome walking “library”. If you now develop a specific primer directed against your gene of interest (GSP1, etc.), the combination of these two primers in the four libraries should result, following PCR, in the producion of varying regions of your specific gene. After these PCR products are isolated, cloned and sequenced, a comparison of the sequences will allow you to complete the sequence of your entire gene.
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