Lecture 10-11

Question 1

Bacterial expression vectors:

Promoter sequence

Answer 1

(for the correct sequence)
Promoter­ allows efficient transcription of the
inserted gene (drives transcription), therefore RNA
binds

Question 2

Bacterial expression vectors:

Operator sequence

Answer 2

(for the correct sequence)
Operator­ permits regulation through a specific
repressor that will bind to it

Question 3

Bacterial expression vectors:

Polylinker

Answer 3

Polylinker with unique sites for several restriction
endonucleases (ie. cloning sites­insert gene of interest)
○ Polylinker/MCS­ site of interaction for gene of
interest

Question 4

Bacterial expression vectors:

Transcription termination sequence

Answer 4

can improve stability
of mRNA and protein yield (minimizes cellular energy
drain)

Question 5

Bacterial expression vectors:

Selectable genetic marker

Answer 5

(eg. antibiotic resistance)­

selectable marker allows selection of cells containing the recombinant expression vector

Question 6

Bacterial expression vectors:

Origin of replication

Answer 6

a sequence of DNA at which replication is initiated

Question 7

Bacterial expression vectors:

Gene encoding repressor

Answer 7

Gene encoding repressor that binds to O and regulates P

Question 8

Bacterial expression vectors:

Ribosome binding site (RBS)

Answer 8

provides sequence signals

required for efficient translation of mRNA derived from the inserted gene.

Question 9

Bacterial expression vectors:

pGEX- differences?

Answer 9

Ptac promoter: modified promoter to increase binding of RNA polymerase
lacq: mutated lacI gene with higher levels of repressor control of protein expression

Encodes N ­terminal GST for affinity tag and PreScission for protease cleavage

Question 10

Bacterial expression vectors:

pET

Answer 10

Pt7: promoter for T7 RNA polymerase

Add IPTG:
-Lac repressor inactivated on both plasmid and bacterial genomic DNA
-T7 RNA pol is produced by host cell RNA pol
-T7 RNA polymerase can now transcribe the gene of interest
= over expression of protein from target gene

Question 11

Bacterial expression vectors:
Lac operon?

If no lactose is present?

Answer 11

Lac operon: c​ollection of three genes required for lactose transport and metabolism in E.coli
● Adjacent structural genes: lacZ (beta­galactosidase), lacY (lactose permease) and lacA (thiogalactoside transacetylase)

If no lactose is present: lac genes are turned off, no enzyme production, therefore energy is saved
Genetic switch turns genes off and on=regulation

Question 12

Regulation of expression using Lac repressor:

Control mechanism

Answer 12

● Lac repressor (regulatory protein) encoded by lacI gene
● LacI gene is constitutively expressed
● In absence of lactose, Lac repressor binds tightly to
operator sequence
● Lac repressor interferes with binding of RNA polymerase
to promoter
● →prevents gene transcription

Question 13

Regulation of expression using Lac repressor:

1. in presence of lactose (or allolactose) ?
2. Consequence?

Answer 13

1. Lac repressor cannot bind to operator sequence due to
   structural/conformational changes caused by lactose
   binding to the repressor protein
2. Lac repressor no longer interferes with binding of RNA polymerase to promoter
   →lacZ, Y and A are transcribed

Question 14

Induction of protein expression (function of IPTG)

Answer 14

Using the lac repressor in vitro
● can exploit this system for controlled regulation of protein
expression in vitro
● Need to provide:
○ lacI gene plus promoter and operator sequence (expression vector)
● IPTG: bind to lac repressor protein and release from operator, therefore ability to control transcription to regulate lac repressor protein

Question 15

Optimising translation of proteins

Answer 15

● Need effective translation of proteins from mRNA
transcripts
● If codon usage in the over­expressed protein differs
significantly from the host cell, problems may arise during
protein expression
● Therefore, the expression system needs to maximise the
translation of proteins from this mRNA

Question 16

Correct processing of proteins:

Answer 16

● Many proteins require the formation of disulphide (S­S)
bonds to fold properly
● Without S­S bond, proteins may be degraded or precipitate in the cell

Question 17

Problem:
● expression of proteins occurs in cytoplasm of E.coli
● cytoplasm is a reducing environment that can prevent S­S
bond formation

Answer 17

Solution:
Use E.coli strains engineered with mutations in:
● gor­ glutathione reductase
● trxB­ thioredoxin reductase
These altered E.coli strains can promote disulphide bond formation and correct folding in the cytoplasm

Question 18

Affinity tags for protein purification

1. What is the basis?
2. Used for what?
3. Where is it placed?

Answer 18

1. Proteins have structural domains: fold independently and have discrete functions
2. Can add domains to proteins that can be used to facilitate solubility, folding and enhance purification
3. Affinity tag placed where it will not interfere

Question 19

Affinity tags for protein purification

1. 6 X His tag
2. MBP
3. GST

Answer 19

1. 6x His tag: interacts with cobalt and nickel ions
2. MBP: interacts with maltose (amylose)
3. GST: interacts with reduced glutathione