PCR amplification and overexpression of the positive regulatory gene chlR in Streptomyces venezuelae
J. L. CLAYTON – BROWN
The polymerase chain reaction (PCR) is a technique used in the amplification of DNA which utilises thermal stable polymerase, Thermus aquaticus (Taq) and primers which aid in the annealing of the chosen DNA strand, producing numerous replications through a cycle of appropriate temperature changes (Lorenz, 2012). Developed in 1983 by Dr. Kary Banks Mullis, PCRs ability to quantitate transcription levels of specific genes has revolutionised research and the understanding of gene function (Bustin, 2000) in its many applications, including the ability to: detect DNA polymorphs and point mutations (Orita et al., 1989), amplify specific genes for the construction of overexpression vectors (Liang et al., 2015), and recognising bacterial (Hill, 1996) and viral (Holodniy, 1994) pathogens. Advances within PCR have only broadened the spectrum of its implementations, with new techniques such as Quantative PCR, and Inverse PCR yielding new insights into once misunderstood areas of molecular biology (Jain and Varadarajan, 2013).
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ChlR is a cluster-associated transcriptional activator consisting of 987 base pairs within the putative CHL biosynthetic operon, predicated to encode the only positive regulator responsible for the initiation of production of chloramphenicol (CHL) (Fernández-Martínez, et al., 2014); with the usance of PCRs cloning capacity, it is predicted that the introduction of a plasmid capable of overexpression of the chlR gene will result in amplified activity of the CHL biosynthetic gene cluster.
DNA amplification by means of PCR often requires a high fidelity taq polymerase within the PCR mixture to minimise mutations (McInerney et al., 2014). The chlR DNA fragment was inserted into the vector pIJ10257  prior to PCR. The final reaction mixture consisted of the following: 10μl 5X Colorless GoTaq® Reaction Buffer, 2μl PCR Nucleotide Mix (10mM each dNTP), 5μl Apra_BamHI_F primer, 5μl Apra_BamHI_R primer, 2μl purified chlR chromosomal DNA, 5μl Dimethyl sulfoxide (D MSO), 20μl Nuclease-Free water, and 1μl GoTaq® G2 DNA Polymerase, equating to a total reaction volume of 50μl within a sterile, nuclease-free PCR tube, labelled as group 2.
The appropriate annealing temperature was set accordingly with the melting temperature (Tm) of the hybridising portion of the primer. The extension temperature was calculated upon the approximation of 1 minute per every 1kb of required amplified DNA. The resulting thermal cycle was applied as such: an initial cycle at 95°C for 5 min (denaturation), 95°C for 30 secs (secondary denaturation), 56°C for 30 secs (annealing), 72°C for 90 secs (extension), repeated for 30 cycles from secondary denaturation. The final extension temperature was 72°C for a period of 5 mins (holding temperature 10°C). Gel electrophoresis (GE) was preformed using a 0.8% polysaccharide agarose gel within a Tris/Borate/EDTA (TBE) buffer and inserted into the 2nd column.
Gels were removed from the gel box and inspected underneath a UV light. No band of DNA was visible within column 2; the molecular weight ladder and chromosomal DNA within columns 3, 13, and 16 from other accompanying PCR (run simultaneously under the same conditions previously described) were observable. When compared to the molecular ladder, successful DNA fragments indicated a base pair (bp) length of approximately 1000bp, in correlation with the 987bp of the chlR regulatory sequence, an indication that the inserted DNA is present.
Fig.1 0.08% agarose gel exhibiting the DNA ladder and S. veneuzlae chromosomal DNA within column 3, 13, and 16. Column 3 shows an excessive quanity of DNA, an indication overproportionate amount of template DNA were added during procedure. The absence of DNA within column 2 (indiciated in red) evinces the failed PCR described in this paper.
The absence of DNA within column 2 demonstrated the failure to obtain a PCR product. As each component was correctly incorporated, other aspects must be adjusted to result in an adequate amount of DNA cloning. There are several alterations implementable to increase the likelihood of success within the reaction, firstly being the redesign of appropriate primers as the most crucial component for successful amplification of the reaction (Dieffenbach et al., 1993); analysis based software for enhancing the specificity of the primers without compromising their sensitivity can be implemented, with programs such as Primer3 and QuantPrime offering the possibilities of designing internal oligonucleotides alongside primer pairs, and the optimisation of these primer pair designs enabling specificity evaluation, respectively (Noguera et al., 2014).
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If the primers present correctly, changes to the temperature cycle should next be ensured. A decrease in the annealing temperature has previously shown to reduce the risk of unspecific binding and preferential amplification (Sipos et al., 2007). A final modification to the protocol is to adjust the number of PCR cycles, as this change can influence aspects of the reaction; a low PCR cycle number may provide accurate estimation of bacterial richness and a decrease of PCR errors (Ahn et al., 2012), whilst an increase in cycles can improve fluorescent intensity of some dyes (SYBR® Green I) (Ramakers et al., 2003).
Electroporation is a common method of transformation concerning plasmids, involving a brief high-voltage pulse which renders the membrane pores to transiently open and allow the subsequent uptake of DNA into the host cell (Pigac and Schrempf, 1995); an associated example is an electrotransformed Escherichia coli bacteriumwith a cloned, overexpressed chlR gene.
In order to clarify correct insertion and amplification of the correct sequence, the DNA must be sequenced. The most common method of DNA sequencing for cloned PCR products is the Sanger sequence, which technique lies in the use of chain-terminating nucleotides (Sanger, et al., 1977). Once clarified, the replicative vector can then be transferred to S. venezuelae via coagulation from the E. coli, transferrable due to the origin of transfer (oriT) within the vector (Mazodier et al., 1989).
It Is expected that an overexpression of the chlR gene would result in elevated levels of the encoded transcription factor protein, initiating increased transcription of the CHL biosynthetic cluster and producing a higher chloramphenicol yield; this would be observable through analysation via High Performance Liquid Chromatography, a sensitive method appropriate for gene expression analysis (Sivakumaran, et al., 2003).Recent research  has strongly indicated that the constitutive expression of chlR effects the overall expression of the speculated, proceeding genes within the cluster, confirming chlR’s role as a transcriptional activator (Fernández-Martínez et al., 2014).
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