MS LECTURE 2 VECTORS

Question 1

Expression vectors have to?

Answer 1

Facilitate expression (transcription and translation) of the inserted open reading frame

Question 2

Expression vectors have to contain

Answer 2

a promoter region upstream of the inserted gene
product (to facilitate transcription)
* have to contain other elements required for proper transcription and translation of the gene (e.g. transcription termination sites, translation
initiation sites)
These have to be matched with the cell system they are used for.

Question 3

pET system

Answer 3

Inducible bacterial expression plasmid

Question 4

T7 promoter under the control of

Answer 4

lac operator

Question 5

T7 being under lac operator control ensures that

Answer 5

expression of our inserted gene product can be regulated.

Question 6

T7 promoter is usually repressed by

Answer 6

lac repressor protein, but can be de-repressed by adding IPTG (isopropyl-beta-D-thiogalactopyranoside).

Question 7

Why use inducible promoters?

Answer 7

Some recombinant proteins are toxic to the cells and
kill them or stop their growth.
Protein quality can suffer, e.g. by aggregation and/or
degradation when large amounts of protein are
produced in a cell.
Making protein costs energy, regulation ensures protein isn’t produced unnecessarily

Question 8

Induce protein production (by adding IPTG) when cells are in

Answer 8

early/mid log growth phase.

Question 9

Protein production is monitored by

Answer 9

determining the optical density (OD) of the bacterial
culture with a spectrophotometer (the more bacteria, the cloudier the
culture).

Question 10

Regulation of protein expression in the lac operon: without inducer

Answer 10

Repressor binds to operator, preventing transcription of lac operon

Question 11

Regulation of protein expression in the lac operon with the presence of an inducer

Answer 11

Allolactose binds to repressor and prevents its binding to the operator, meaning that the genes can be transcribed. In the lab IPTG is added which is a non-hydrolysable analogue of allolactose, to de-repress genes under the control of the lac operator

Question 12

Regulatory elements can be used to

Answer 12

drive expression of other genes e.g. GOI in pET

Question 13

Genes involved in lactose metabolism

Answer 13

B-galactosidase, permease, transacetylase

Question 14

T7 promoter is transcribed by

Answer 14

T7 RNA polymerase, a a gene encoded by the T7 phage. High transcription rate. Means the T7 RNA polymerase also needs to be introduced into E. coli as it does not naturally occur in these cells

Question 15

Genetically modified E. coli strain used for protein expression

Answer 15

E. coli strain BL21/DE3

Question 16

E coli strain BL21/DE3

Answer 16

Inducible expression of T7 RNA polymerase (under control of lac operon).
T7 RNA polymerase gene and elements of the lac operon have been introduced into the bacterial chromosome.
Addition of IPTG de-represses both, the T7 RNA polymerase and the gene of interest in pET plasmid.
BL21/DE3 strain is also deficient in ompT and lon proteases.

Question 17

Inducible bacterial expression vectors (like pET) in BL21/DE3 E.coli absence of inducer

Answer 17

In the absence of IPTG, repressor binds to lac operator and transcription is switched off. Lac operator controls
expression of T7 polymerase in BL21/DE3 E.coli strain and expression of insulin gene in pRSET.

Question 18

Inducible bacterial expression vectors (like pET) in BL21/DE3 E.coli presence of inducer

Answer 18

IPTG binds to repressor and renders it unable to
binds to lac operator. Transcription is unblocked. T7 polymerase is expressed in BL21/DE3 strain and can access T7 promoter in pRSET vector. Lac operator in pRSET is also unblocked by IPTG.

Question 19

Expression of proteins in mammalian cells

Answer 19

We can introduce plasmid vectors into mammalian cells
through:
- transfection (e.g. Calcium phosphate-mediated or
liposome-mediated)
Or
- transduction with viral vectors.

Question 20

Features of mammalian expression vectors

Answer 20

You have to be able to amplify these plasmids in E.coli before use, so they still
contain a bacterial replication origin and antibiotic resistance gene (here
Kanamycin). Vectors that work in two different systems are called ‘shuttle
vectors’ (e.g. E.coli/mammalian or E.coli/Yeast).
SV40 origin allows episomal replication in cells lines that contain Simian Virus
40 (SV40) large T antigen (e.g. HEK293T cells). This leads to increased
plasmid numbers in the mammalian cells and therefore higher expression levels.
Gene of interest under control of mammalian or viral promoter (e.g. CMV
promoter from Cytomegalovirus, cellular EF-1a or Ubiquitin C promoters), the
promoter should mediate high level constitutive expression.
SV40 poly A: transcription termination site, adds poly-A tail to mRNA

Question 21

Different vectors for different purposes;
For expressing fusion proteins (tag sequence contained in vector) :

Answer 21

1. Containing affinity or epitope tags: His, GST, Ha, c-myc, flag…
   The epitope tags allow you to purify and/or detect your protein
   (e.g. affinity purification or immunoprecipitation using antibodies
   directed against epitope tag, Western Blot).
2. Containing fluorescent protein tags (e.g. green fluorescent
   protein, GFP). Allows you to visualise your fusion protein in cells,
   organisms).

Question 22

Different vectors for different purposes
Inducible expression:

Answer 22

Tet on/Tet off system (expression only in the presence/absence of
tetracyclin). Similar idea to lac operon/lac promoter system, but
engineered for mammalian system.

Question 23

Affinity tags:

Answer 23

6x Histidine (His-tag)
Glutathione-S-transferase (GST-tag)
—->
Affinity-tagged
proteins can be
purified via affinity
chromatography

Question 24

Epitope tags:

Answer 24

Epitope-tagged
proteins detected with
antibodies raised to
these epitopes. They
can also be
‘immunoprecipitated’
using these antibodies
coupled to a solid
matrix.

Question 25

GFP-tagged proteins

Answer 25

Green fluorescent protein (GFP)
first isolated from Jellyfish Aequorea
victoria in 1962.

Question 26

Tet-on/Tet-off system (inducible expression in mammalian cells)

Answer 26

Based on E.coli system: Tet repressor protein (tetR) negatively
regulates genes of the tetracycline-resistance operon in the tn10
transposon (similar principle to lac operon).

Question 27

Engineered for use in mammalian cells: tetR

Answer 27

CMV (CytomegaloVirus) promoter under the control of several tet
operator elements (tetracycline responsive element, TRE)
-fusion to a transactivation domain turns TetR into a tetracyclinecontrolled transactivator (tTA).
- systems have been developed where addition of tetracycline (or
the related doxycycline) switches transcription on (tet-on) or off
(tet-off).

Question 28

Tet off:

Answer 28

Regulatory protein (tTA) is fusion
protein of aa 1-207 of tetR and the
transactivation domain of the HSV
transcription factor VP16. This
fusion turns TetR into a
transcriptional activator. Binding of
tetracycline to tTA prevents its
binding to the promoter and
therefore turns off gene expression.