15 April

• Genomic libraries

  Collections of DNA fragments that represent the entire genome of an organism
• Genomic libraries

  • Useful for identifying and isolating genes of interest
  • Allow the study of gene structure and regulation

  
• Constructing a genomic library

  1. Isolate genomic DNA

  2. Fragment DNA

  3. Clone DNA fragments into vectors

  4. Transform vectors into host cells

  
• Expression vectors

  Designed to allow the experimenter to control the expression of cloned genes

  
• Expression vectors

  • Native promoter of cloned gene may work poorly in new host
  • Overproduction of foreign proteins may damage host cell

  
• Regulating expression of cloned genes

  1. Typically at the transcriptional level

  2. Require strong promoters that bind RNA polymerase efficiently

  
• Example of strong promoter

  • Bacteriophage T7 promoter and T7 RNA polymerase

  
• Using T7 expression system

  1. Cloned genes placed under control of T7 promoter

  2. T7 RNA polymerase gene integrated into host chromosome under lac promoter

  3. Transcription of cloned genes switched on by lac inducer IPTG

  
• T7 expression system

  • T7 RNA polymerase highly active, uses most RNA precursors
  • Limits transcription to cloned genes, host genes not transcribed
  • Yields primarily the protein of interest

  
• Ensuring efficient translation of mRNA

  1. Require ribosome-binding site and start codon on mRNA

  2. Adjust codon usage to match host tRNA levels

  
• Cloning genes from eukaryotic mRNA

  1. Isolate mRNA

  2. Convert to cDNA by RT-PCR

  3. Insert cDNA into expression vector with bacterial promoter and RBS

  
• Artificial gene synthesis

  Advantages: no introns, can include promoters and regulatory sequences, codon bias can be optimized

  
• Purifying overexpressed proteins

  1. Fusion proteins with carrier proteins

  2. Carrier protein chosen to avoid inclusion bodies and be easy to purify

  3. Protease cleavage site between carrier and target protein

  
• Fusion proteins

  • Simplify purification of target protein
  • Carrier protein can contain signal sequence to enable secretion

  
• Site-directed mutagenesis

  Using synthetic DNA and cloning techniques to introduce mutations at precise locations in a gene

  
• Site-directed mutagenesis

  1. Use PCR with primers containing desired mutation

  2. Exchange mutated PCR product into target gene using recombineering

  
• Primers or probes for use in the polymerase chain reaction (PCR) and hybridization

  Obtained in the same manner as DNA oligonucleotides
• Using PCR to obtain a gene with a specific mutation

  1. Determine position of desired change

  2. If target gene is part of E. coli chromosome or cloned into vector

  3. PCR product or oligonucleotide containing desired mutation can be exchanged through recombineering

  
• This scheme allows any base pair in a specific gene to be changed
• Site-directed mutagenesis

  1. Progeny bacteria screened through PCR and Sanger sequencing to detect those carrying the mutation

  2. Mutated gene expressed to produce protein with altered amino acid sequence

  
• Applications of site-directed mutagenesis

  • Manipulate proteins to test functional importance of specific amino acids
  • Change specific amino acid in enzyme active site to compare with wild-type
  • Insert mutant enzyme into host unable to make original enzyme to measure activity of mutant alone
  • Link enzyme's activity, interactions to specific amino acids

  
• Cassette mutagenesis

  Using synthetic DNA fragments to mutate DNA and replace sections of a gene of interest

  
• Cassette mutagenesis to make insertion mutations

  1. Cassette containing antibiotic resistance gene inserted into cloned gene

  2. Linearized plasmid transformed into host, antibiotic resistance selected for

  3. Cassette inserted by homologous recombination between mutated gene on plasmid and wild-type gene on chromosome

  
• Knockout mutations

  • Disruption of gene function, similar to insertion mutations by transposons but experimenter chooses gene to mutate
  • Viable only in haploid organisms if disrupted gene is nonessential

  
• Reporter gene fusion

  Coding sequence from one source fused to regulatory region from another to form hybrid gene, used to study regulation of gene expression

  
• Properties of reporter genes

  • Encode easily detectable protein
  • Used to report presence/absence of genetic element or to study gene expression when fused to other genes/promoters

  
• Reporter genes


  • lacZ (E. coli beta-galactosidase)
  • Green fluorescent protein (GFP)

  
• Operon fusions


  Coding sequence retains own translational start, fused to transcriptional signals of another gene

  
• Protein fusions


  Genes encoding two different proteins fused together, share transcriptional and translational signals
• Using gene fusions to study gene regulation

  1. Regulatory region of gene of interest fused to reporter gene coding sequence

  2. Reporter expression assayed under different conditions to determine how target gene is regulated

  
• Using gene fusions to test regulatory genes

  Regulatory gene mutations introduced into cells carrying gene fusions, expression measured and compared to cells lacking mutations

  
• Other uses of gene fusions


  • Joining easily purified proteins to proteins of interest to aid purification