Laboratory: General Information

Question 1

What are plasmid vectors?
(7)

Answer 1

Tiny extra-chromosomal pieces of double stranded circular DNA

Found in bacterial cells

Couple thousand base-pairs long

Many copies per cell

Autonomously self replicating (have own origin or replication)

Carry genes that convey antibiotic resistance

Have restriction enzyme cut sites

Question 2

What type of genes do plasmids usually have?

Answer 2

Antibiotic resistance genes

Question 3

Plasmids have restriction enzyme cut sites what is this useful for?

Answer 3

The uptake of foreign DNA

Question 4

What is an operon system?

Answer 4

Genes that work together and are regulated together

Question 5

Give three examples of operons

Answer 5

Lac operon
Trp operon
Arabinose operon

Question 6

List the different parts
(6)

Answer 6

Bacterial origin of replication

Selectable marker e.g. Ampicillin resistance

Multiple cloning site

Strong inducible, promoter with control e.g. Lac

Methods of inhibiting protein reduction e.g. LacI

Method of purifying protein (GST, His fusions)

Question 7

TE buffer-Tris EDTA buffer
(4)

Answer 7

It is a buffer used to protect DNA from degradation

It is used to store DNA

The Tris, CI pH 8 in buffer is to maintain the PH of solution in alkaline

The EDTA in TE buffer is a chelating agent. It chelates Ca and Mg ions which are cofactors required by DNAses

Question 8

Write a note on the arabinose operon
(4)

Answer 8

The arabinose operon uses both positive and negative control

Contains three different genes (ara A, ara B and ara D) which code for three enzymes needed to convert arabinose to a usable form

A fourth gene codes for a protein (ara C) that acts to regulate the structural genes

They encode metabolic enzymes for breaking down arabinose

Question 9

What is the ampicillin resistance gene called?

Answer 9

bla

Question 10

What is a pGLO plasmid made of?
(4)

Answer 10

Green fluorescent protein gene

araC gene (regulates GFP transcription)

bla gene (ampicillin resistance gene) ( produces beta-lactamase enzyme)

ori (origin of replication - allows plasmid replication)

Question 11

What does the bla gene produce?

Answer 11

Beta lactamase
Which induces ampicillin resistance

Question 12

What does the bla gene produce?

Answer 12

Beta lactamase
Which induces ampicillin resistance

Question 13

How does transformation take place?

Answer 13

Treat bacteria with calcium chloride and then undergo heat-shock @42 degrees celsius

Question 14

How does transformation via Calcium chloride work?

Answer 14

The positive charge of calcium chloride ions neutralises:
- the negative charge of DNA phosphates
- the negative charge of membrane phospholipids
- slows down the fluidity of the membrane and cell wall

Question 15

How does heat shock work?

Answer 15

Activates the heat shock proteins and seals the cell membrane and traps plasmid inside

Question 16

What do you do after plasmid transformation?

Answer 16

Recovery in highly rich nutrient broth (SOC medium)

This allows the bacteria to recover from the transformation procedure and allows the b-lactamase gene to produce ampicillin to facilitate the selection process

Question 17

How have we modified the araC gene?
(3)

Answer 17

AraC is now the regulator for GFP

The genes of the ara operator (B, A and D) have been replaced with the gene for GFP

Now the addition of arabinose turns the araC operon on which thusly expresses GFP

Question 18

What does the araC gene usually do?

Answer 18

The araC gene or arabinose operator is responsible for breaking down arabinose into usable parts

The B, A and D genes of araC are responsible for making enzymes

Question 19

What happens before and after you introduce arabinose to the modified araC gene?
(3)

Answer 19

Without arabinose the araC repressor blocks transcription of the gene and therefor GFP

In the presence of arabinose, arabinose binds repressor and changes its conformation

The altered repressor leaves DNA and now RNA polymerase can perform transcription and express the GFP

Question 20

What is electroporation?

Answer 20

Exposing cells to high voltage in the presence of plasmid DNA and low salt conditions

Allows for uptake of plasmid