MI2011: Biotech Flashcards
Vectors
tools for inserting DNA into cells for sequencing or expression
What are two types of vectors that are manipulated
Bacteriophage vectors and Plasmid vectors
Bacteriophage vectors
- can use in vitro packaging in which you add your DNA, phage heads and tails and enzymes
- results in infectious virus particles capable of injecting your DNA into a susceptible host cell
Plasmid vectors
- use restriction endonucleases to cut DNA at specific sties, use DNA ligase to re-join
- T-vector cloning for PCR products
- Induce competency in bacterial cells, uptake the plasmid by transformation
Shuttle vectors
can replicate and be stably maintained in 2 or more unrelated hosts
Expression vectors
include regulatory sequences to switch on and off expression of inserted genes
Phage vectors
phage genome allows for holding larger amounts of DNA than a plasmid
Cosmid vectors
specialized plasmids that use phage virions
Artificial chromosomes
- bacterial artificial chromosomes are engineered from conjugative plasmids
- yeast artificial chromosomes, replicate in yeast like normal chromosomes and allow a large DNA fragment insertion
Mutagenesis
- mutates a gene at a specific point
- hybridises a mismatched oligonucleotide
- oligo acts as primer for synthesis
- new DNA strand contains mutant gene
- Useful for: protein engineering; constructs proteins with new properties
Recombinant expression: Protein expression
Insert genes into vectors which have promotors that result in expression of the gene. Result: production of specific protein
Transfection
Introduce plasmid into eukaryotic cells for expression of protein
- transient (plasmid not integrated into chromosome)
- stable (plasmid integrates)
Describe how a colony containing a plasmid with an insert is selected
Blue-white screening
List some examples of therapeutics
- Immunomodulators
- Hormones (insulin)
- Enzymes
- Growth Factors
- Vaccines
- Gene therapy
What bacteria is typically used as a cloning host in eukaryotes
Saccharomyces cerevisiae
