BGM1004/L06 Recombinant DNA Technology II

Question 1

Why don’t all plasmids contain the desired DNA insert?

Answer 1

Some relegate back to their original form as they are all cut by the same enzyme

Question 2

What gene is disrupted by insertion of DNA fragments into polylinker?

Answer 2

lacZ (beta-galactosidase)

Question 3

What colour do bacteria containing the pUC18 insert produce?

Answer 3

Blue

Question 4

Give 2 ways of checking if bacteria contain the desired insert.

Answer 4

Hybridisation to ssDNA probe
PCR using specific primers
Screen for expression
Attach probe to sequence

Question 5

How does CRISPR-CAS9 guarantee that offspring will inherit the desired trait?

Answer 5

Animal has 2 identical chromosomes

Question 6

How does CRISPR-CAS9 give 2 identical chromosomes? (2)

Answer 6

Guide RNA directs CAS9 to cut wildtype DNA
Replaced by altered gene in existing chromosomes

Question 7

Give 3 uses for recombinant DNA.

Answer 7

Induce expression in host
Recover and manipulate further
Recover and transfer to different cell type

Question 8

Why are proteins directly expressed after DNA recombination? (2)

Answer 8

For purification
To investigate function

Question 9

Why are modified versions of a protein expressed? (2)

Answer 9

To change properties
To investigate fine detail

Question 10

What do artificial inducers (IPTG) do?

Answer 10

Stimulate transcription

Question 11

What do artificial inducers bind to?

Answer 11

Lacl repressor protein

Question 12

What does a Lacl repressor do?

Answer 12

Prevent expression of cloned gene

Question 13

How can we ensure that the inserts are oriented correctly?

Answer 13

Use two different restriction enzymes

Question 14

What are introns?

Answer 14

Non-coding DNA

Question 15

When are introns removed?

Answer 15

Maturation of mRNA

Question 16

Why can certain proteins not be made in bacterial cells?

Answer 16

Splicing does not occur in bacterial cells

Question 17

What occurs as a result of genomic DNA ligation into an expression vector?

Answer 17

Inactive protein expression

Question 18

What kind of DNA contains the coding sequence with no introns?

Answer 18

cDNA

Question 19

Give a typical bacterial expression system.

Answer 19

E. coli
B. subtilis

Question 20

Give 2 advantages to bacterial expression systems.

Answer 20

Simple cells
Short generation times
Large product yield
Low cost

Question 21

Give 2 disadvantages to bacterial expression systems.

Answer 21

Eukaryotic proteins can fail to fold correctly
Proteins can be toxic to cell
No post-translational modifications

Question 22

Give 2 advantages to yeast expression systems.

Answer 22

Simple unicellular eukaryote
Resembles mammalian cells
Quick and cheap
Post-translational modifications

Question 23

Give a disadvantage to yeast expression systems.

Answer 23

Contains proteases
Post-translational modifications may differ from mammalian cells

Question 24

Give 2 advantages to insect cell expression systems.

Answer 24

Active - high-level expression
Correct folding of proteins
Post-translational modifications
Cheaper than mammalian cell culture

Question 25

Give a disadvantage to insect cell expression systems.

Answer 25

Post-translational modifications may differ from mammalian cells

Question 26

Where is insulin made?

Answer 26

Beta-cells in Islets of Langerhans (pancreas)

Question 27

Give 2 disadvantages to bovine/porcine insulin for diabetes treatment.

Answer 27

Side effects
Difficult to purify
Possible contamination

Question 28

Describe the structure of insulin.

Answer 28

2 polypeptide chains linked by disulfide bonds
RNA transcribed into chain A&B then joined

Question 29

What is unfolded insulin called?

Answer 29

Proinsulin

Question 30

What is removed from proinsulin to form insulin?

Answer 30

Connecting peptide

Question 31

How many introns does the proinsulin gene contain?

Answer 31

2

Question 32

How are introns removed when producing recombinant human insulin?

Answer 32

mRNA is reverse-transcribed to produce proinsulin cDNA

Question 33

What antibodies block HIV infection in vitro?

Answer 33

Anti-CD4

Question 34

How was the hypothesis that CD4 is the cellular receptor for HIV tested? What was the outcome?

Answer 34

Expressing recombinant CD4 in cells which don’t usually express CD4
These cells became infected