I. DNA & RNA | 19. Generation and application of recombinant DNA, reporter- and expression vectors

Question 1

I. Basics
1. Describe the genome of bacterium

Answer 1

Bacterial DNA contains a circular chromosome and plasmids

Question 2

I. Basics
2. Characteristics and role of plasmids

Answer 2

• Plasmids are circular, double-stranded (dsDNA) molecule that are separate from chromosomal DNA
• Each plasmid contain 1 - 2 protein coding genes
• It is replicated independently of cell division

Question 3

II. DNA Cloning
1. Definition of DNA cloning.

Answer 3

• In DNA cloning, recombinant DNA molcules are formed in vitro by inserting DNA fragments into vector DNA molecules
• The recombinant DNA molecules are then introduced into host cells, where they replicate, producing large numbers of recombinant DNA molecules

Question 4

II. DNA Cloning
2. Which protein is the first medicament generated by recombinant technology?

Answer 4

INSULIN

Question 5

II. DNA Cloning
3. Where can recombinant protein be synthesized?

Answer 5

• It can be synthesized using pro- or eukaryotic cells

Question 6

II. DNA cloning
4. Definition of DNA-construct/ recombinant DNA

Answer 6

Recombinant DNA molecules are formed in vitro by inserting DNA fragments into vector DNA molecules

Question 7

II. DNA cloning
5. What is the basic scheme of DNA cloning?

Answer 7

A. Vector + DNA fragment = recombinant RNA
B. Replication of recombinant DNA within host cells
C. Isolation, sequencing and manipulation of purified DNA fragment

Question 8

II. DNA cloning
6. What are the 4 steps of Generation of DNA-construct?

Answer 8

a. On a gene, there are 2 sites which are recognition site of restriction endonuclease A and recognition site of restriction endonuclease B
b. Type 2 Restriction enzymes (endonucleases) typically cut DNA at specific 4- to 8- bp palindromic sequences, producing defined fragments that often have self-complementary single-stranded tails (sticky ends)
c. Two restriction fragments with complementary ends can be joined to form a recombinant DNA molecule
=> The gene is inserted into a vector

Question 9

II. DNA cloning
7. How is Vector-insert construction incorporated in competent bacterial cells?

Answer 9

a. Cells can be treated chemically or electrically
1. Chemical way: Cells together with the construct (in the presence of Ca2+ ions) are incubated on ice. This leads to formation of pores in membrane. Therefore, then under the heat shock (42 degree C for 45 sec), DNA enters the cells
2. Electrical way: bacteria are treated with electricity (~2000 V, very short time period – couple of milliseconds). Finally, DNA enter the cells

b. We can use agar plate (solid growth medium) with bacteria or placing one colony in liquid growth medium for protein synthesis

Question 10

II. DNA cloning
8. How do we purify recombinant protein after treating bacterial cells?

Answer 10

Purification of recombinant protein by using affinity-chormatography
a. Separation: target protein is bound on the column containing specific ligands for protien of interest.
b. Elution: change of conformation: target protein dissociates

Question 11

II. DNA cloning
9. What are the 3 functional regions of E.coli cloning vector?

Answer 11

1/ An origin of replication which is also promotor
- mRNA synthesis is active or can be activated
2/ A drug-resistance gene
- Selection of bacteria
3/ A site where a DNA fragment can be inserted (gene of other protein)
- Transformed cells carrying a vector grow into colonies on selection medium
- E.g, glutathionine-S-transferase

Question 12

III. Application of DNA cloning
1. What the structure of expression vector?

Answer 12

A vector consist of
- a CMV promotor
- a poly-A tail
- a cloning site containing restriction recognition sites
- Ampicillin resistance site for bacterial selection

Question 13

III. Application of DNA cloning
2. What are the role and example of expression vector?

Answer 13

• For analyzing protein coding region
• E.g, pcDNA 3.1 (plasmid cloning DNA)

Question 14

III. Application of DNA cloning
3. What is the structure of reporter vector?

Answer 14

A vector consist of
- a poly-A tail
- cloning sites containing restriction recognition sites
- Ampicillin resistance site for bacterial selection
- Luciferase enzyme coding gene

Question 15

III. Application of DNA cloning
4. What are the role and example of reporter vector?

Answer 15

1. Role:
   - Analyze regulatory regions such as promotors (how weak or how strong)
2. E.g, pGL3

Question 16

III. Application of DNA cloning
5. Why is it called a reporter gene?

Answer 16

Amount / concentration of product can be precisely measured

Question 17

III. Application of DNA cloning
6. How do we analyze gene expression with reporter gene?

Answer 17

1. Polymorphisms in the 5’ region (or introns) can modulate the activity of transcription.
2. Luciferase enzyme coding gene produce luciferase which can convert luciferin to oxiluciferin with emission of light
3. By determining light intensity emitting from luciferase reaction, we can analyze how strong or how weak the promotor is