Cloning Vectors
Why is a small size vector desirable?
-easy manipulation, high copy number, less vulnerable to damage, easier transformation and more insert per ug of plasmid
what percentage of cells take up plasmid DNA with E.coli?
0.01%
Why is pBR322 a good vector? 2 reasons
1. A few restriction sites which fall within antibiotic resistance genes - selection for recombinants 2. High copy number- 15 per cell
What are the 5 desirable characteristics of a cloning vector?
1.small size 2.selectable markers 3. range of restriction sites for ligation 4.positive selection/identification 5. high copy number
How big can plasmids be?
1kbp to 200kbp
How many selectable markers does pBR322 have? what are they?
2- ampicillin and tetracycline
How many antibiotic resistance genes does the vector RP4 contain?
3
what are the nucleic acids molecule in bacteriophage surrounded by?
A protein coat
What is X-gal? why is it good for selection?
A substrate for B-galactosidase- produces a colour change when broken down by enzyme (turns blue)
What is IPTG?
An artificial inducer of the lac operon inducing production of B-galatosidase
Why do we use mutant E.Coli with other half of lacZ gene?
As a selectable marker- both parts of enzyme can be produced if a complete pUC8 plasmid is taken up by the E.coli
What is the head and tail structure typically?
Bacteriophage lambda
What is the cloning vector pBR322 cut with?
BamH1
What is the enzyme to be cloned into pBR322 cut with?why?
BamH1 - to give the same sticky ends
How are replica plates used for pBR322?
Before placing E.coli colonies on tetracycline medium, create a complete replica plate of the colonies (maintain position of colonies on a new agar plate) this means it is clear to see which colonies can grow on the tetracycline plate due to their positions - can identify recombinants on the replica plate
what does the BR stand for in pBR322?
Bolovar and Rodriquez
What are cloning vectors derived from M13 used for?
DNA sequencing
Since pUC8 only has lacZ' and not the other half of the lac Z gene to produce full B-galactosidasde what do we use?
E.coli mutants with the other half of the lacZ gene
How do we distinguish between cells that have taken up a plasmid or not with pBR322 and E.coli?
E.coli plated out on ampicillin - only cells containing cirular pBR322 (recombinant OR NOT) will be resistant and grow
What is the main vector we use?
E.coli, its plasmids and bacteriophage
To produce the protein coded for by a gene of interest what must occur?
Grow an e.coli containing a recombinant DNA molecule on a Petri dish and grow a colony
There are 2 types of bacteriophage commonly- what are these?
Head and tail & filamentous
What is unique about plasmid replication?
It is autonomous
What is the most common filamentous bacteriophage used?
M13
After killing all the recombinant cells with pBR322 cloned DNA, how do we therefore get our clones without killing them?
Making a replica plate
What does the lacZ' gene code for?
Part of B-galactosidase (not full protein)
How do we recognise E.coli which have taken up a pUC8 (recombinant or not)?
Plate out E.coli on agar with ampicillin
What can we use to distinguish between E.coli which has taken. up a recombinant pUC8 or a complete?
Plate out on IPTG and X-gal
What restriction sites for pBR322 have?
Pst1, EcoR1 , BamH1 and Hind111
If the pUC8 taken up by E.coli contains a cloned insert (recombinant) what occurs?
The lac Z' gene is disrupted by cloned DNA, so half of the proteins for enzyme B-galactosidase will NOT be produced and the enzyme will not work
What does the genetic engineer put into pUC8 for selection?
The lacZ' gene
what is the difference between pUC8 and pUC9?
The order of restriction sites is reversed in pUC9
What kind of colour change does non- recombinant pUC8 e.coli give when plated out on x-gal? why?
The turn blue as the pUC8 plasmid is complete and so lacZ' gene is expressed producing half the proteins for B-galactosidase and the E.coli mutants produce the other half- B-galatodisdase is produced and breaks X-gal down into blue product
Once pBr322 and DNA has been cut- what is the next step?
They are mixed in a suitable buffer with T4 ligase and ATP
what is unique about the family of pUC plasmids?
They come in pairs e.g pUC8 and pUC9
What kind of colour change does recombinant pUC8 e.coli give when plated out on x-gal? why?
They remain white as the cloned DNA disrupts the lacZ' gene so half of the proteins for the enzyme B-galactosidase are not produced so X-gal is not broken down
Why is positive selection desirable?
To distinguish the cells with RECOMBINANT plasmids
Once a colony containing recombinants with gene of interest is grown on a petri dish, what must then occur to obtain DNA?
Use this colony to inoculate a liquid culture and grow overnight, then purify the DNA
What do we have to do to natural plasmid?
We have to modify them from their natural form to make them more convenient to isolate DNA
What kind of restriction sites does pUC8 have? where did they come from?
a cluster engineered into the lacZ gene from the lac operon
When pBR322 what can be added to prevent cell ligation?
alkaline phosphatase- removal of 5' phosphate
what does the complete (not recombinant) pUC8 have? (for selection)
ampicillin resistance and produces part of proteins required for B-galactosidase
What kind of nucleic acid is in bacteriophage? Which kind is generally used by genetic engineers?
can be RNA or DNA but bacteriophage with DNA genomes is generally used
Where is the BamH1 site that we have cloned into in pBR322?
cloned into BamH1 site in pBR322 which is within tetracycline resistance gene
What are cloning vectors used for?
cloning fragments of DNA
What are cloning vectors derived from lambda used for?
cloning large pieces of DNA
Why is a selectable marker desirable?
distinguish plasmids containing cells from non containing (usually antibiotic resistance)
What kind of genome does bacteriophage lambda have? and how big is the genome?
double stranded DNA genome of 50kbp
Why is a range of restriction sites desirable?
for ligating DNA into the vector and cloning fragments can be produced by various enzymes
What does the cluster of restriction sites in lacZ of pUC8 allow?
fragments cut with different enzymes i.e Pst1 and BamH1
How can antibiotic resistance be used as a selectable marker?
grow on medium of antibiotic - only cells with the plasmids containing genes for that specific antibiotic resistance will grow
If the plasmid is smaller it will have a... ?
higher copy number
After treating E.coli with cacl2 what are the next steps?
incubate with ligation products and heat shock to transform cell
What do we do in order to find which pBR322 plasmids have the correct recombinants?
introduce into bacterial cells and use positive selection
Why is the order of restriction sites reversed in pUC9?
it enables cloned DNA fragments to be turned around
whats the most common head and tail bacteriophage used?
lambda
Cloning produces what?
many mg of a selected DNA molecule
Why is a high copy number desirable?
more plasmid DNA per cell
What are cloning plasmids derived from?
naturally occurring plasmids in bacteria or bacteriophage
How many antibiotic resistance genes does pUC8 have? What are they?
one- ampicillin resistance
What are the 2 plasmid cloning vectors you look at?
pBR322 and pUC
what does the p stand for in plasmid vector names?
plasmid
in essence what is cloning?
purifying and amplifying a gene of interest
When DNA is cloned into pUC8, what changes for selection?
recombinants have ampicillin resistance but do not produce proteins for B-galactosidase
Due to the position of the site we cloned into in pBR322, we can use insertional inactivation to distinguish recombinants how?
recombinants will NOT be resistant to tetracycline as this gene has been disrupted - grow on plate of tetracycline - cells that CAN grow are NOT recombinant
What kind of genome does bacteriophage M13 have? and how big is the genome?
single stranded genome of about 10kbp
is pUC8 bigger or smaller than pBR322? how big is it?
smaller- 2750bp
How do we make E.coli cells competent to take up pBR322 ligation products?
treated with cold cacl2
What will the products of ligation between pBR322 and DNA give?
un ligated vectors, self ligated vectors, vectors containing recombinant genes
How do we distinguish between recombinant pBR322 plasmids and self ligated ones?
using insertional inactivation
What are bacteriophages?
viruses that infect bacteria
