Week 10 - Mutagenesis and protein production

Question 1

What is protein engineering?

Answer 1

Targeted modification of protein sequence, used to decrease, increase or create novel function in proteins

Question 2

Site-directed mutagenesis (SDM)

Answer 2

Targeting of specific nucleotides within specific genes, is a technique that provide a lot of control as a single nucleotide can be removed or added to cause cahnges

Question 3

Methods for SDM

Answer 3

PCR methods
Synthetic gene method
M13-directed mutagenesis

Question 4

M13 directed mutagenesis

Answer 4

A gene is selected and a short matching oilgo is synthesized this is then mutated, this is annealed to the selected gene, then converted into DS DNA by polymerase

Question 5

PCR SDM 1

Answer 5

1. methylate DNA with Dam methylase
2. Denature methylated template and anneal divergent mutagenic primers
3. PCR amplifies the entire plasmid with DNA polymerase lacking proof reading
4. Original methylated DNA is digested
5. Unmethylated PCR product is not digested

Question 6

PCR SDM splicing by overlap extension

Answer 6

1. mutation introduced via primers in the first PCR reactions
2. Gene to be mutated is amplified in 2 part by PCR
3. PCR products overlap and can be used to generate a full length copy of the mutated gene
4. final step involves amplification of the entire sequence

Question 7

Artificial gene synthesis

Answer 7

Gene is assembled with overlapping nucleotides, several mutations can be introduced at different points, Gaps filled in and DNA strands joined by DNA polymerase and DNA ligase then cloned

Question 8

what is needed for Recombinant protein production

Answer 8

Needs a host like E. coli, the gene must be inserted into an expression vector

Question 9

What is in an expression vector?

Answer 9

P - Promoter
R - Ribosome binding site
T - Terminator

These are used to control gene expression

Question 10

List some promoters used in expression vectors

Answer 10

Lac - widely used
tac - similar to lac
T7 - also uses lac for more effective control

Question 11

list some facts about Lac promoters

Answer 11

Lac is induced by IPTG
Lac is represseb by Lacl binding
Lacl can be displaced from lac promoter by addition of inducing reagent such as IPTG
adition of IPTG results in increased expression from lac promoter

Question 12

Tac promoter

Answer 12

Used in combonation with lac and induced by IPTG, provides greater control of expression

Question 13

T7 promoter

Answer 13

Requires strains containing T7 RNA polymerase gene cloed under control of lac promoter, addition of IPTG induces T7 RNA polmerase expression which controls expression on plasmid