Recombinant DNA - Bacterial Transformation Flashcards
Describe what a plasmid is and their functions
A plasmid is a circular form of DNA, typically found in the cytoplasm of a prokaryotic cell or in plant/fungi eukaryotic cells.
Plasmids function independently, physically separate from chromosomal DNA.
The main purpose of plasmids are to carry survival genes; antibiotic, heavy metal - resistance
In lab settings, plasmids are used for pharmaceutical purposes, such as production of insulin as plasmids can act as a vehicle to transfer genes through bacterial transformation
Explain the uses of plasmids
Plasmids have multiple uses as a result of their easily manipulative structure
Plasmids are used as tools to manipulate and clone genes through the use of vectors
Different forms of plasmids have different functions; virulence plasmids, degradative plasmids or fertility plasmids
Define what a restriction enzyme is, and provide an example
A restriction enzyme is used to remove artificial restriction site to replace with foreign DNA.
Restriction enzymes cut ‘sticky ends,’ or ‘blunt ends,’ at specific sites.
Restriction enzymes can also protect bacteria from viruses by invading foreign DNA.
An example of a restriction enzyme is restriction endonuclease.
Define what a bacterial transformation is
Bacterial transformation is the process in which a plasmid is modified, where a restriction enzyme cuts at a restriction site and is replaced with a modified gene through ligase enzyme. The plasmid is then transferred to other bacterium through cell to cell contact in a process known as conjugation. This is possible as conjugative plasmids are self-transmissible and contains conjugation genes (tra genes)
Define what a vector is
a DNA molecule (often plasmid or virus) that is used as a vehicle to carry a particular DNA segment into a host cell as part of a cloning or recombinant DNA technique
Understand how restriction enzymes are specific and can produce sticky or blunt ends
The restriction enzymes cut in different locations as different restriction enzymes have specific sites which they are programmed to cut. The method in which restriction enzymes cut can leave either sticky or blunt ends. A sticky end is when the restriction enzyme cuts on one opposite end of complementary strand producing nucleotides without complementary bases. This makes it easier for the replacement DNA to attach to the complementary base pairs.
Describe how a human gene can be used to mass produce insulin using E.coli
1) DNA and vector is isolated and insulin is amplified via PCR
2) The insulin gene and the plasmid vector is both cut by restriction endonuclease enzyme at recognition sites - restriction endonuclease cleaves sugar phosphate backbone to form sticky ends
3) Ligation of vector; target gene and vector have complementary overhangs, via complementary base pairing they join together and are spliced through DNA ligase to form recombinant construct
Ligase joins vector and gene through a covalent phosphodiester bond
4) Selection and expression; recombinant construct is introduced through heat shock to appropriate cell/organism - antibiotic selection is used to identify successful constructs
List requirements for gene transfer and production of insulin
- Isolated human insulin gene
- Susceptible bacteria with plasmids
- Restriction enzymes
- DNA ligase
- Host cells
- Large fermentation tanks
State examples of pharmaceutical drugs produced by bacterial transformation.
- Streptomycin
- Tetracycline
- Vancomycin
- Insulin
