Lecture 15

Question 1

What is cloning

Answer 1

A sequence of DNA is inserted into a plasmid vector. This vector can then be put into a cell

Question 2

Why do we want to clone genes ?

Answer 2

The aim of gene coining is to obtain isolated and purified copies of specific gene sequences

Question 3

Cloned genes allow us to:

Answer 3

• determine the sequence of the gene and protein
• obtain leads into the function of the gene
• manipulate the gene e.g.
  - mutate the gene
  - insert the gene into the cells or tissue of an organism
  - make large amounts of protein

Question 4

Question 5

Definition of a clone:

Answer 5

A large number of identical cells or molecules with a single ancestral cell or molecule

Question 6

Definition of a DNA cloning vector:

Answer 6

A carrier DNA molecule that allows attached DNA to be replicated in a cell e.g. plasmids and viruses

Question 7

Definition for a restricition enzyme

Answer 7

An enzyme that recognises and cleaves DNA at specific sequences e.g EcoRI cleaves at the palindrome GAATTC

Question 8

What is DNA ligase

Answer 8

An enzyme that can covalently link DNA molecules

Question 9

What are plasmids ?

Answer 9

Double stranded DNA molecules that replicate in cells independently of the host chromosome. They are inherited by daughter cells and are non-essential for growth

Question 10

Why are plasmids idea cloning vectors

Answer 10

• well characterised (sequence and function of genes knows)
• small and easy to purify and manipulate

Question 11

Useful features of plasmid cloning vectors:

Answer 11

• sequence known
• an origin of replication
• a selectable marker
• unique restriction enzyme cleavage sites
• easy methods to screen recombinants
• often high copy number

Question 12

What makes plasmids ideal cloning vectors?

Answer 12

• well characterised (sequence and function of genes known)
• small and easy to purify and manipulate

Question 13

What 3 things must a plasmid vector contain?

Answer 13

• an origin of replication
• a selectable marker
• unique restriction enzyme cleavage sites

Question 14

4 steps involved in cloning a piece of DNA

Answer 14

1) RESTRICTION: the plasmid vector and the DNA fragment of interest must be digested with a restriction enzyme to produce ‘ sticky ends’

2) LIGATION: the DNA fragment of interest must be lighted into a plasmid vector

3) TRANSFORMATION: the lighted plasmid vector must be transformed into a bacterial host (usually E.coli)

4) SELECTION: bacterial colonies carrying the plasmid of the DNA fragment of interest must be selected from those that do not

Question 15

Making a plasmid clone

Question 16

1. Cutting DNA - sticky ends and 2. Pasting DNA - ligation

Answer 16

-cut DNA at a specific sequence

We take our plasmid DNA and we cut with the restriction enzyme to make those sticky overhangs
Cut the DNA we are going to clone and cut it with the same restriction enzyme to make complementary sticky ends
Stick ends together using ligase enzyme

Question 17

Introducing plasmids into bacteria - two key methods

Answer 17

To make many copies of the gene, it needs to get inside a ‘host’. Plasmids commonly put into E.coli bacteria via:

• transformation
• electroporation

Question 18

Process of transformation to introduce plasmids into bacteria

Answer 18

• cells are made ‘competent’ to take up DNA by treating with ice-cold CaCl2, mixed up with DNA and then heat-shocked at 42 degrees

Question 19

How does the process of electroporation work when introducing plasmids into bacteria

Answer 19

• cells are made ‘competent’ too take up DNA by treatment with an electric field

Question 20

A mix of restricted plasmid vectors and DNA of interest that has been treated with ligase is transferred into bacterial cells, the ligation mix will be (below), but what different populations of E.coli cells will result from the transformation?

Answer 20

Ligation mix will be:
- uncut (relegated) vector (plasmid rejoins with itself or may not be cut)
- unligated insert
- cut vector
- vector plus insert
- bacteria picks up no DNA

Question 21

Two ways pUC allows for selecting and screening recombinant plasmids

Answer 21

• antibiotic resistance (Beta lactamase cleaves ampicillin)
• colour change

Question 22

Using pUC how are we screening for antibiotic resistance ?

Answer 22

• only bacteria with uncut and the correct insert in vector will survive

Question 23

PUC19 vector - screening by colour change

Answer 23

• puc19 vector has a gene for colour change

Question 24

What does lacZ a gene encode?

Answer 24

B-galactosidase a-peptide

Question 25

Do we put the whole lacZ gene o our cloning vector? Why?

Answer 25

No - would make it inefficient and too big We just put on a the alpha gene, which encodes the alpha peptide of the B-galactrosidase gene

Question 26

What does B-galactose need to be able to cleanse X-gal and form a blue product

Answer 26

Alpha peptide

Question 27

Answer 27

Question 28

Answer 28

When we use the pUC vector, we have to use a bacteria that is compatable with using the pUC vector, so the bacteria that we use has a deletion in the lac Z alpha gene but producing the rest of the lac Z gene. - so it can’t produce alpha peptide but it can produce B-galactosidase missing alpha peptide - this means if the B-gal is to be activated, it means it needs the alpha peptide from pUC vector which compliments the non-functioning B-galactosidase

Question 29

What happens when there is no DNA insert in put vector

Answer 29

Question 30

What happens when there is a DNA insert in the LacZ a gene?

Answer 30

Question 31

How to screen by antibiotic resistance and colour change

Answer 31

Plate onto ampicillin to select bacteria with an intact plasmid and on Xgal to select plasmids with an instern - blue colonies have active B-galacrtosidase enzyme so no insert - white colonies have inactive B-galactosidase so must have a distracted a-fragment and therefore have a DNA insert - white colonies are clones that have arisen from a single bacterium carrying a single insert and are the clones of interest

Question 32

Will be in exam

Answer 32

Question 33

What is a multiple cloning site?

Answer 33

- within the lacZ gene - string of base pairs - loads of restriction enzyme recognition sites - means you can cut and open up vector using heaps of different restriction sites an enzymes

Question 34

Where is the lacl gene?

Answer 34

On the pUC vector

Question 35

What does the lacl gene encode?

Answer 35

The repressor (controls the ability to turn on the lacZ gene, IPTG must be added to induce the lacZ gene)

Question 36

What must be added to induce the lacZ gene? How does it work?

Answer 36

IPTG must be added to induce the lacZ gene IPTG binds the lac repressor - acts like allolactose - however it is not broken down by B-gal, thus permantly able to switch on the lacZ gene

Question 37

Need to use pUC vector with a compatible E.coli strain e.g DH5-alpha - what makes it compatible?

Answer 37

- lacks the lacZ alpha gene - bit produces the rest - can’t produce alpha peptide - B-gal is produces with a missing alpha peptide

Question 38

We want to know if the DNA has been clones into the lacZ alpha gene - if it has been its white cos it can’t produce the alpha peptide and thus it can’t cleave X-gal