2. Genetic modification of microbes Flashcards
Types of genetic variation
Mutation
Recombination
Random mutagenesis
Random mutations caused by exposure to a mutagenic agent
Types of mutagens
Physical: radiations
Chemical
Biological: transposons
Types of mutagenesis by recombination
Sexual reproduction (eukaryotes)
Protoplast fusion
Protoplast fusion
Fusing paternal and maternal cells to create a mutant
Genetic engineering
Introducing mutations using DNA technology
Main steps of recombinant DNA technology
Genetic engineering
Incorporation of recombinant DNA into the cell
Selection of clones
Methods for introduction of DNA into cells
Transformation
Electroporation
Conjugation
Transduction
Origin of replication
Particular sequence in a genome at which replication starts
Expression in cis
A cis gene regulates expression of genes on the same DNA
Expression in trans
A trans gene regulates expression of genes on separate DNA
Example of regulation in cis
Operons
Example of regulation in trans
Plasmids
Process for introducing a directed deletion in a genome
Deleting a fragment of the coding sequence in a separate plasmid
Opening the target DNA and plasmid with the same restriction enzyme
Ligating the ends of the plasmid with target DNA
Process for introducing a point mutation in a genome
Synthesis of two sets of DNA primers containing the mutation in its center
Extension with DNA polymerase
Ligation of the two PCR products
Extension
Introduction by allelic exchange
Advantages of using eukaryotes as hosts for expression of human DNA
Their genetic machinery is similar to ours
How do you check if your vector was inserted correctly?
You include antibiotic cassettes or metabolic markers into the vector
Metabolic markers
Genes that encode an enzyme that is essential for growth of the organism in the culture medium
Process of selection with X-gal
Vector carries part of the gene on each end of the MCS
If the insert was introduced, that gene will be broken (because the insert will be inside it) and the enzyme will not be expressed (white colonies)
If the insert was not introduced, the enzyme is expressed (blue colonies)
Expression vector
Plasmid designed for protein expression in cells. It contains a specific gene and uses the cell’s mechanisms for protein synthesis
Features common to all vectors
Origin of replication
Selectable marker
Multicloning site
Elements necessary for protein expression
Transcription: promoter, termination sequence
Translation: RBS, start codon, termination codon
Inducible promoter
Promoter whose activity is induced by the presence or absence of a particular substance in the medium
Utility of peptide tags
Ensure that protein is not degraded or misfolded
Facilitates detection and purification of product
Linker sequences
Short sequences included in the peptide tag to allow us to remove the tags
Methods for purification using tags
Anti-tag antibodies bind the tag
If the tag is maltose binding protein, we use maltose to bind the tag
Glutathione S transferase (GST) tagged proteins are purified with glutathione
Polyhistidine tails can be detected and retained in a column
Periplasmic or external secretion signal
Signal peptide that induces or improves product secretion
Most commonly used types of yeast
Saccharomyces cerevisiae
Pichia pastoris
Types of cloning vectors derived from yeast
YEP
YAC
