Lecture 15 Flashcards
What is cloning
A sequence of DNA is inserted into a plasmid vector. This vector can then be put into a cell
Why do we want to clone genes ?
The aim of gene coining is to obtain isolated and purified copies of specific gene sequences
Cloned genes allow us to:
- 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
Definition of a clone:
A large number of identical cells or molecules with a single ancestral cell or molecule
Definition of a DNA cloning vector:
A carrier DNA molecule that allows attached DNA to be replicated in a cell e.g. plasmids and viruses
Definition for a restricition enzyme
An enzyme that recognises and cleaves DNA at specific sequences e.g EcoRI cleaves at the palindrome GAATTC
What is DNA ligase
An enzyme that can covalently link DNA molecules
What are plasmids ?
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
Why are plasmids idea cloning vectors
- well characterised (sequence and function of genes knows)
- small and easy to purify and manipulate
Useful features of plasmid cloning vectors:
- sequence known
- an origin of replication
- a selectable marker
- unique restriction enzyme cleavage sites
- easy methods to screen recombinants
- often high copy number
What makes plasmids ideal cloning vectors?
- well characterised (sequence and function of genes known)
- small and easy to purify and manipulate
What 3 things must a plasmid vector contain?
- an origin of replication
- a selectable marker
- unique restriction enzyme cleavage sites
4 steps involved in cloning a piece of DNA
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
Making a plasmid clone
- Cutting DNA - sticky ends and 2. Pasting DNA - ligation
-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
Introducing plasmids into bacteria - two key methods
To make many copies of the gene, it needs to get inside a ‘host’. Plasmids commonly put into E.coli bacteria via:
- transformation
- electroporation
Process of transformation to introduce plasmids into bacteria
- 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
How does the process of electroporation work when introducing plasmids into bacteria
- cells are made ‘competent’ too take up DNA by treatment with an electric field
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?
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
Two ways pUC allows for selecting and screening recombinant plasmids
- antibiotic resistance (Beta lactamase cleaves ampicillin)
- colour change
Using pUC how are we screening for antibiotic resistance ?
- only bacteria with uncut and the correct insert in vector will survive
PUC19 vector - screening by colour change
- puc19 vector has a gene for colour change
What does lacZ a gene encode?
B-galactosidase a-peptide
Do we put the whole lacZ gene o our cloning vector? Why?
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
What does B-galactose need to be able to cleanse X-gal and form a blue product
Alpha peptide
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
What happens when there is no DNA insert in put vector
What happens when there is a DNA insert in the LacZ a gene?
How to screen by antibiotic resistance and colour change
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
Will be in exam
What is a multiple cloning site?
- 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
Where is the lacl gene?
On the pUC vector
What does the lacl gene encode?
The repressor (controls the ability to turn on the lacZ gene, IPTG must be added to induce the lacZ gene)
What must be added to induce the lacZ gene? How does it work?
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
Need to use pUC vector with a compatible E.coli strain e.g DH5-alpha - what makes it compatible?
- lacks the lacZ alpha gene - bit produces the rest
- can’t produce alpha peptide
- B-gal is produces with a missing alpha peptide
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
