![]() Second, the gene-of-interest is then packaged into an infectious virion via the expressed viral particles. First, a cell is transfected with numerous plasmids encoding the gene-of-interest along with various viral particles. Transduction, on the other hand, is a multi-step process. 7 For transformation (into bacteria) or transfection (into mammalian cells), the cell will express the recombinant protein. Liposomes are also employed to deliver DNA to cells (Figure 2). 5 In either case, the plasmids encoding the gene-of-interest passively enter the cell through its membrane, which has been temporarily compromised (i.e., “holy”) via calcium phosphate or electroporation. The viral vector flanks the gene-of-interest by viral sequences, which eventually result in the gene being inserted into the host chromosome permanently.Īfter cloning, genes encoding for the gene-of-interest are introduced into the host system, either transiently by a cDNA plasmid (i.e., transformation, transfection) or permanently by viral infection (i.e., transduction). The antibiotic resistant gene enables the selection of cells carrying the plasmid in antibiotic-based media. It also often has a sequence at the N- or C-terminus encoding a fusion tag for downstream protein purification or identification. 5,6 An expression vector is a cDNA plasmid that includes a promoter sequence and an antibiotic resistant gene. The synthesized gene is then cloned into an expression or viral vector. The efficiency of protein translation is improved by optimizing the nucleic acid sequences that reflect the codon bias (due to the varying tRNA pools) of the chosen expression system. For example, four codons (CTT, CTC, CTA, CTG) will be translated to the amino acid residue, leucine. ![]() The nucleic acid sequence is important to consider since one amino acid is encoded by different 3-nucleic acid sequences called codons. The first step of making a recombinant protein is synthesizing the gene-of-interest. ![]()
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