MS LECTURE 1 VECTORS

Question 1

Cells: We can use prokaryotic cells (E.coli) or
eukaryotic cells (Yeast, mammalian cell lines) as

Answer 1

‘bioreactors’ to produce the protein of interest.

Question 2

Gene of interest: We first need to isolate the gene
for the protein of interest: use

Answer 2

PCR to amplify from
cDNA of relevant cell type.

Question 3

Vector: use recombinant DNA technology to insert
gene of interest into an appropriate vector. The
vector is needed to

Answer 3

shuttle the gene of interest into
the cells, and to facilitate amplification of the GOI
and/or expression of the protein in the cells.

Question 4

Why are different vector systems used?

Answer 4

To incorporate larger inserts

Question 5

The main vector systems

Answer 5

1.Bacterial plasmid <3 to 10kb
2.Lambda phage vector up to 25kb
3.Cosmid vector 30 to 45kb
4.BAC 100 to 300kb

Question 6

Plasmids are

Answer 6

extrachromosomal, circular genetic material with their own
origin of replication.

Question 7

Plasmid vectors are derived from

Answer 7

naturally occurring bacterial Rplasmids (carrying antibiotic resistance genes)

Question 8

Plasmid vectors have been modified for use in recombinant DNA technology

Answer 8

• smaller,
• higher copy number
• multiple cloning site (or polylinker)
• no oriT (for transfer between cells)

Question 9

In plasmid vectors Ampicillin resistance gene
allows

Answer 9

you to isolate bacteria that have taken up
the plasmid)

Question 10

In plasmid vectors the multiple cloning site/ polylinker contains several unique restriction sites allowing

Answer 10

You to insert DNA fragment

Question 11

In plasmid vectors the lacZ gene for B-galactosidase allows for

Answer 11

blue-white screening (is insert present or not)

Question 12

The 2 types of plasmid vectors?

Answer 12

Simple cloning vectors
Expression vectors

Question 13

Simple cloning vectors

Answer 13

For amplification of recombinant plasmid
(including the cloned gene, ‘insert’) in E.coli.
Cloned gene of interest can be sequenced
from these plasmids.
Good ‘storage’ for PCR fragments

Question 14

Expression vectors

Answer 14

• allow you to actually transcribe and translate the cloned gene in
  either E.coli or mammalian cells.
• have to contain a promoter region upstream of the inserted gene
  product.
• have to contain other elements required for proper transcription
  and translation of the gene (e.g. transcription termination sites,
  translation initiation sites)

Question 15

How is cDNA made?

Answer 15

Synthesising DNA that is complementary to mRNA

Question 16

Limitations for plasmid vectors

Answer 16

Can only take relatively small DNA inserts: -up to 10kb
Taken up into E.coli by the process of transformation, which is
relatively inefficient:
- Maybe only 1 in every 2000 plasmids gets taken up by E.coli
- depends on size, purity and integrity of the plasmid
- preparation of cells: incubate in CaCl2 and heat-shock at 42˚C

Question 17

Lambda phage (an E.coli virus): Bacteriophages are viruses that infect bacteria. They essentially consist of ?

Answer 17

Genomic nucleic acid surrounded by a protein coat

Question 18

Lambda phage is much better at entering E. coli than?

Answer 18

Naked plasma DNA

Question 19

Lambda phage for vector use

Answer 19

Remove non-essential genes from bacteriophage genome and insert
your gene of interest instead (can take larger insert, up to 25kB).
Bacteriophage genome will be packaged into phage head
Infect E.coli with recombinant phage, which will then replicate (and
amplify your gene of interest)

Question 20

Lambda phage 2 life cycles

Answer 20

Lytic and Lysogenic cycles

Question 21

Lambda phage lytic life cycle

Answer 21

1. Phage attaches to host cell and injects DNA
2. Phage DNA circularises and enters lytic or lysogenic
3. New phage DNA and proteins are synthesised and assembled into virions
4. Cell lyses, releasing phage virions

Question 22

Lambda phage lysogenic life cycle

Answer 22

1. Phage attaches to host cell and injects DNA
2. Phage DNA circularises and enters lytic or lysogenic
3. Phage DNA integrates within the bacterial chromosome by recombination, becoming a prophage
4. Lysogenic bacterium reproduces normally
5. Occasionally, the prophage may excise from the bacterial chromosome by another recombination event, initiating a lytic cycle

Question 23

Generating recombinant lambda phage

Answer 23

Prepare phage
Extract DNA from phage
Cut DNA with restriction enzyme
Ligate your EcoR1- digested insert with phage arms
COS (cohesive) ends mediate concatemer formation
Concatemers can be in vitro packaged into phages to infect new host cells:
DNA between two Cos ends is packaged into one phage head.
Plate on bacterial lawn and observe plaques (phage present and lyses E.coli)

Question 24

Cosmid vectors are ?

Answer 24

A mix between a plasmid and a phage vector (up to 45 kB insert)

Question 25

Packaging of linearised recombinant Cosmids into empty  phage particle in vitro, mediated by

Answer 25

cos sites.

Question 26

Following infection of bacteria, replication of Cosmids within E.coli cell like

Answer 26

plasmid rather than lambda phage (via ori).

Question 27

Cosmid vector also contains

Answer 27

SV40 replication origin that allows plasmid propagation in mammalian cells lines.

Question 28

Cos sides incorporated into plasmid vector. XbaI digest

Answer 28

generates linearised plasmid with Cos sides at either end (resembling phage DNA)

Question 29

Phage and other viral vectors have the advantage of being

Answer 29

very efficient at introducing the DNA into cells (approaching 100%).

Question 30

Introducing DNA into cells is called?

Answer 30

Transduction

Question 31

Transduction is based on

Answer 31

the natural route of infection (or a modified version thereof).

Question 32

Examples of transduction

Answer 32

Baculovirus vectors used for transduction of insect cell lines Lentiviral/Retroviral vectors used for transduction of mammalian cell lines

Question 33

Bacterial Artificial Chromosomes (BACs) * Based on F-plasmid:

Answer 33

– Naturally occurring E.coli plasmid, “Fertility” factor – Conjugative transfer between E.coli cells – Extrachromosomal or integrated into host genome

Question 34

BAC clones can contain DNA fragments of up to 300kb length (used for genome projects). Are introduced into E.coli using

Answer 34

electroporation (higher efficiency than chemical transformation/heat shock). But low copy number (usually just 1 copy per cell).