Genetic control of metabolism

Question 1

Wild type/strain

Answer 1

Strain of microbes that has been collected directly from its natural environment

Question 2

Wild type/strain phenotype

Answer 2

Typical form of a species as it occurs in nature

Question 3

How can the pure strains of these wild types be isolated?

Answer 3

Then wild types can be cultured in an enriched nutrient medium

Question 4

Why are wild types very useful in industry?

Answer 4

They can produce a great number and variety of different metabolites

Question 5

Improvements that may still need to be made to the wild strain to make them suitable for use in industry -

Answer 5

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

Question 6

2 techniques used to improve microorganisms -

Answer 6

Mutagenesis and recombinant DNA technology

Question 7

Mutagenesis -

Answer 7

The creation of mutants by inducing mutations.

Question 8

How are mutations induced?

Answer 8

Exposure of organisms to mutagenic agents

Question 9

Examples of mutagenic agents

Answer 9

UV light other forms of radiation (X rays)

Or mutagenic chemicals like dioxin

Question 10

Problems with mutagenesis

Answer 10

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

Question 11

How can the improved strain mutated state be ensured?

Answer 11

Used in an industrial fermenter and will be constantly monitored.

Question 12

Recombinant DNA technology

Answer 12

Transfer of gene sequences from one organism to another and even from one species to another.

Question 13

Improvements made by Recombinant DNA technology -

Answer 13

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

Question 14

Process of recombinant DNA creation

Answer 14

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

Question 15

Restriction endonuclease enzyme is used to

Answer 15

Cut specific gene out of chromosome of the donor organism.

Cut open the bacterial plasmid that are to receive the genetic information.

Question 16

How does restriction endonuclease enzyme work?

Answer 16

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.

Question 17

Sticky ends

Answer 17

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

Question 18

DNA ligase use in DNA recombination

Answer 18

It is the same enzyme that seals the gene into the plasmid to form a recombinant plasmid

Question 19

Recombinant DNA vector

Answer 19

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.

Question 20

Requirements for a plasmid to be an effective vector -

Answer 20

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.

Question 21

Restriction site of the vector -

Answer 21

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

Question 22

Selectable markers

Answer 22

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.

Question 23

Origin of replication

Answer 23

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.

Question 24

Why is it good that many genes are expressed?

Answer 24

More products can be made by fewer cells.

Question 25

What does the regulatory sequences that control gene expression and origin of self replication allow?

Answer 25

Self replication of the plasmid/artificial chromosome

Question 26

Why are artificial chromosomes preferable to plasmids?

Answer 26

When larger fragments of foreign DNA are required to be inserted.

Question 27

Limitations of prokaryotic hosts

Answer 27

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

Question 28

How can the incorrect folding of polypeptides be avoided?

Answer 28

Using recombinant yeast cells as opposed to bacterial hosts for plant or animal recombinant DNA.