Screening of Bacterial Recombinants: Strategies and Preventing False Positives

IntroductionComplete decoding of complex eukaryotic genomes is a prerequisite for understanding varied gene functions.Gene silencing (point mutations, gene deletions, etc), sub cellular localization of proteins, gene expression pattern analysis by promoter activity assay, structure-function analysis, and in vitro or in vivo biochemical assays (Hartley et al., 2000;Curtis & Grossniklaus, 2003;Earley et al., 2006) are some of the approaches followed for understanding gene functions.Typically, all the above approaches require the cloning of target genes with or without selective mutations, or cloning their promoter fragments into specialized vectors for further characterization.While the traditional approach for constructing expression cassettes that is based on the restriction enzyme/ligase cloning method is laborious and time consuming, the process is often hampered by length of the gene of interest, GC content of the gene, toxicity of the gene product to the expressing host and lack of relevant restriction sites for cloning purposes.All these factors render the production of expression constructs a significant technical obstacle for large-scale functional gene analysis.After generating successful cloning/expression constructs, several steps followed are screening high number of colonies, avoiding false positive recombinants and requirement of dephosphorylation of vectors in case of single site cloning to ensure the generation of recombinants with rightly oriented gene of interest and to minimize vector background (non-recombinants).Screening for recombinants is one of the most crucial and time-consuming steps in molecular cloning and several approaches available for this purpose include colony PCR screening, blue white screening, screening of recombinants, which have the gene of interest in the MCS region of the cloning vehicle, in such a way that the toxic gene reading frame is interrupted making the toxic gene inactivated upon insertion of any foreign gene; GFP fluorescence vectors wherein upon cloning, the GFP fluorescence disappears, etc.The method for screening of bacterial transformants that carry recombinant plasmid with the gene of interest, has become more rapid and simple by the use of vectors with visually detectable reporter genes. Molecular cloningA recombinant DNA comprises of two entities namely a vector and the gene of interest (GOI).The process of joining vector and any GOI is by making a phosphodiester bond by a www.intechopen.comMolecular Cloning -Selected Applications in Medicine and Biology 4 process called ligation.The ligation reaction is facilitated with the help of T4 DNA ligase in the presence of ATP.If a vector and any target DNA fragments are generated by the action of the same restriction endonuclease, they will join by base-pairing due to the compatibility of their respective ends.Such a construct is then transformed into a prokaryotic cell, where unlimited copies of the construct, an essentially the target DNA sequence is made inside the cell. Steps in molecular cloningThe conventional restriction and ligation cloning protocol involves four major steps namely fragmentation of DNA with restriction endonucleases, ligation of DNA fragments to a plasmid vector, introduction into bacterial cells by transformation and screening and selection of recombinants. Selection and preparation of vector and insertA cloning vehicle, also termed as a vector, can be classified as a carrier carrying a gene to be transferred from one organism to another.Other cloning vectors include plasmids, cosmids, bacteriophage, phagemids and artificial chromosomes.In the early days of producing proteins in E. coli, limitations to transcription initiation were believed to lead to lower protein expression levels (Gralla, 1990).This event resulted in efforts put into construction of expression vectors, which carried strong promoters to enhance mRNA yield and a stable mRNA eventually.The promoters used included phage promoters like T7 and T5, the synthetic promoters tac and trc, and the arabinose inducible araBAD (Trepe, 2006).Additional vectors that were made available included Lambda promoters, PR and PL, (Elvin et al., 1990), rhamnose promoter (Cardona & Valvano, 2005), Trp-lac promoter (Chernajovskyi et al., 1983) etc. Certain promoter variants as seen in the expression vector pAES25 yield the maximum level of soluble active target protein (Broedel & Papciak , 2007).Downstream of each specific promoter, there is a multiple cloning site (MCS) for cloning the gene to be expressed.While the inducible promoters are used to drive the foreign gene expression, the constitutive promoters (Liang et al,., 1999) are used mainly to express the antibiotic expression marker genes for plasmid maintenance.TA cloning vectors (Zhou & Gomez-Sanchez, 2000;Chen et al., 2009) that takes advantage of the well-known propensity of non-proofreading DNA polymerases (e.g., Taq, Tfl, Tth) to add a single 3´-A to PCR products are also employed for cloning large PCR fragments.The proof-reading polymerases lack 5'-3' proofreading activity and are capable of adding adenosine triphosphate residues to the 3' ends of the double stranded PCR product.Such a PCR amplified product can then be cloned in any linearized vector with complementary 3' T overhangs.The GC cloning technology is based on the recent discovery that the above proof-reading enzymes similarly add a single 3´-G to DNA molecules, either during PCR or as a separate G-tailing reaction to any blunt DNA.GC cloning vectors pSMART® GC and pGC™ Blue (commercialized by Lucigen, USA) contain a single 3´-C overhang, which is compatible with the single 3´-G overhang on the inserts.Mead and coworkers (Mead et al., 1991) report cloning of PCR products without any restriction digestion taking advantage of the single 3' deoxyadenylate extension that www.intechopen.com