Genetic control of metabolism Flashcards
Wild type/strain
Strain of microbes that has been collected directly from its natural environment
Wild type/strain phenotype
Typical form of a species as it occurs in nature
How can the pure strains of these wild types be isolated?
Then wild types can be cultured in an enriched nutrient medium
Why are wild types very useful in industry?
They can produce a great number and variety of different metabolites
Improvements that may still need to be made to the wild strain to make them suitable for use in industry -
Produce large quantities of the target compound
Improvement of genetic stability
Improved ability to grow on low cost nutrients
Allow easy harvesting of target compound after fermentation is complete
2 techniques used to improve microorganisms -
Mutagenesis and recombinant DNA technology
Mutagenesis -
The creation of mutants by inducing mutations.
How are mutations induced?
Exposure of organisms to mutagenic agents
Examples of mutagenic agents
UV light other forms of radiation (X rays)
Or mutagenic chemicals like dioxin
Problems with mutagenesis
Even if improved characteristics are obtained, we cannot control what mutation will be.
Furthermore, the mutant strains are often genetically unstable so may undergo reversed mutations and may revert back to the less useful wild type state
How can the improved strain mutated state be ensured?
Used in an industrial fermenter and will be constantly monitored.
Recombinant DNA technology
Transfer of gene sequences from one organism to another and even from one species to another.
Improvements made by Recombinant DNA technology -
Amplify specific metabolic steps in metabolic pathways to increase yield of target compound
Remove inhibitory controls in a pathway to increase yield of target compounds
Cause cells to secrete product into surrounding medium for ease of harvesting
Process of recombinant DNA creation
Identify specific gene in the chromosome, them cut out the required gene via the use of a specific restriction endonuclease enzyme. Removal of plasmid (vector) from a bacterial cell. Cut open plasmid through the use of the same restriction endonuclease enzyme. Specific gene will be inserted into the plasmid via the use of DNA ligase. Place plasmid into a new bacterial cell. The bacteria cell multiples making many copies of the gene
Restriction endonuclease enzyme is used to
Cut specific gene out of chromosome of the donor organism.
Cut open the bacterial plasmid that are to receive the genetic information.
How does restriction endonuclease enzyme work?
Each restriction endonuclease recognises a specific short sequence of DNA bases called a restriction site and cuts the strand in such a way that it can leave sticky ends.
Sticky ends
Produced when nucleotides are cut at different points, these sequences are 4-8 nucleotides in length and are complementary to the other sticky end made by the same enzyme as such they will “stick” together
DNA ligase use in DNA recombination
It is the same enzyme that seals the gene into the plasmid to form a recombinant plasmid
Recombinant DNA vector
A DNA molecule used to carry foreign genetic information into another cell.
Both plasmids and artificial chromosomes are used as vectors during recombinant DNA technology.
They carry DNA from the genome of one organism to another and bring about transformation.
Requirements for a plasmid to be an effective vector -
Restriction sites to insert DNA, selectable marker genes to allow identification of host cells which have taken up the vector or not, an origin of replication for self replication and regulatory sequences to control gene expression.
Restriction site of the vector -
Contains target sequences of DNA where the specific restriction endonuclease cuts, same as the endonuclease used to cut the genes out, as such sticky ends will be complementary to one another and the ligase will be able to combine them - causing the formation of a recombinant plasmid
Selectable markers
Things like antibiotic resistant genes protecting the microorganism from a selective agent that’d normally kill the microorganism. This enables the scientists to determine which plasmids have taken up the recombinant plasmid.
Origin of replication
Where inserted genes, genes for self replication of plasmid DNA and regulatory sequences to control expression or plasmid.
It is the site where DNA replication begins.
Why is it good that many genes are expressed?
More products can be made by fewer cells.
What does the regulatory sequences that control gene expression and origin of self replication allow?
Self replication of the plasmid/artificial chromosome
Why are artificial chromosomes preferable to plasmids?
When larger fragments of foreign DNA are required to be inserted.
Limitations of prokaryotic hosts
If DNA fragments is from eukaryotic organism then it the DNA will contain introns, and in a bacterial cell splicing won’t occur so polypeptide folding folding issues may occur and therefore the synthesis of inactive eukaryotic proteins
How can the incorrect folding of polypeptides be avoided?
Using recombinant yeast cells as opposed to bacterial hosts for plant or animal recombinant DNA.