Genetic Engineering Tools PDF

I. Tools of the Genetic Engineer 12.1 Manipulating DNA: PCR and Nucleic Acid Hybridization 12.2 Molecular Cloning 12.3 Expressing Foreign Genes in Bacteria 12.4 Molecular Methods for Mutagenesis 12.5 Reporter Genes and Gene Fusions  Tools of the Genetic Engineer 12....

I. Tools of the Genetic Engineer 12.1 Manipulating DNA: PCR and Nucleic Acid Hybridization 12.2 Molecular Cloning 12.3 Expressing Foreign Genes in Bacteria 12.4 Molecular Methods for Mutagenesis 12.5 Reporter Genes and Gene Fusions  Tools of the Genetic Engineer 12.2 Molecular Cloning Molecular cloning: Movement of a gene from original source to small and manipulable genetic element (vector) results in recombinant DNA Gene can be manipulated. cloned DNA replicated Research: structure, biochemistry etc. Production of proteins Enzymes for industry Cloning to Biologics a plasmid Production of Gene amplifications DNA DNA vectors for gene therapy etc.  isolation and fragmentation of source DNA Cloning process can be amplified by polymerase chain reaction, synthesized by reverse ¤ transcriptase, or synthetic DNA made in vitro insertion of DNA fragment into cloning vector small, independently replicating genetic elements than can carry and replicate cloned DNA designed to allow insertion of foreign DNA at a restriction site, a specific DNA sequences recognized by Restriction endonucleases/enzymes, resulting in double-stranded breaks with blunt or sticky ends DNA ligase joins/anneals DNA introduction of cloned DNA into host organism Cloning vectors several types and copy number varies use dependent on size of fragment to be cloned and host plasmids (e.g., pUC19) widely used (Figure 12.8) Plasmid selection: contains ampicillin resistance and Cloning efficiency by blue- white color-screening: contains multiple cloning site within lacZ gene encoding lactose- -galactosidase Figure 12.8  Note two key characters of the plasmid: 1. Copy number variation 2. Replication of origin and compatibility (if two type of there plasmids can be in the same cell) klnde of lasmidk I why many ? so ave https://www.gs.washington.edu/academics/courses/manoil/former/41108/lecture/lectureFeb25.pdf Hosts for cloning vectors Figure 12.10 12.3 Expressing Foreign Genes in Bacteria Transcription and translation of cloned genes using expression vectors Expression vectors: allow experimenter to control the expression of cloned genes. important to regulate expression, typically at transcriptional level require strong promoters, (e.g., bacteriophage T7 promoter and T7 RNA polymerase) foreign. T7 expression vectors Cloned genes are placed under control of the T7 promoter. gene for T7 RNA polymerase present and under control of easily regulated system (e.g., lac) (Figure 12.11) BL21 E. coli strains (e.g., BL21(DE3) strain) specially designed to work with pET T7 expression vectors and induction by Isopropyl - ~ d-1-thiogalactopyranoside (IPTG). IPTG is a molecular mimic of ~ allolactose, a lactose metabolite that triggers transcription of the lac operon. - no need of sigma factors Iihe 2inl. Figure 12.11 θ Plasmid map of pET24 Expression vectors must ensure mRNA is efficiently translated. needs appropriate ribosome-binding site (RBS) and start codon Bacterial RBSs must be engineered into vector for eukaryotic gene expression. other adjustments for high-efficiency translation codon usage: related to concentration of appropriate tRNA; one could perform codon optimization according to the host species codon optimization: used to change selected codons to match usage of host; typically done by gene synthesis. important to regulate expression, typically at transcriptional level  Gene cloning Eukaryotic gene clone: typically modified via mRNA, which is easy to isolate because of poly(A) tails and mRNA contains no introns. convert into complementary DNA (cDNA) by RT-PCR bacterial promoters and ribosome- Splicing binding sites used for high-level expression needs appropriate ribosome-binding site (RBS) and start codon Can artificially synthesize entire genes (around 1 RMB per nt) or even a chromosome Promoters and other regulatory sequences can be inserted upstream of coding sequence. Codon bias can be adjusted to adapt to the host species Figure 12.12  Protein stability and purification Recombinant proteins may cause problems to the host cells (e.g., degradation by proteases, toxicity, insoluble inclusions) Host can be modified to increase stability of the recombinant protein. e.g. mutate the lon protease Culture and IPTG induction conditions can be adjusted, e.g. express protein at a low temperature 18 °C Fusion proteins joining target and carrier proteins can simplify purification. fuse two genes linked with a protease cleavage site into one coding sequence Transcription and translation yield single protein purified by methods designed for carrier. Cleavage by protease releases target. Carrier protein does not form inclusions. An example: E. coli maltose-binding protein vector (Figure 12.13) Signal sequence can be added for secretion. 12.5 Reporter Genes and Gene Fusions Coding sequence from reporter is fused with regulatory region from another source. Regulation studied by assaying reporter under different conditions sensed by regulator. RNA transcription Regulatory Gene X region Regulatory region Protein X Regulator rexpl aemFirst widely used was lacZ from Escherichia coli o아 -galactosidase. X-gal cleaved to give blue color. examples: lacZ, luciferase, GFP genes Protein X lacZ Protein Y GFP Protein Z Luciferase GFP Protein Y Protein fusions: Genes encoding two proteins are fused to share the same transcriptional and translational start and stop and yield one hybrid polypeptide.  Can be fused to green Can be fused to other genes or fluorescent protein (GFP) other gene promoters to study to study the location of a ~ gene expression protein (Figure 12.16) ~ Protein X GFP Figure 12.17