Download Cloning Using Plasmid Vectors

Cloning Using Plasmid
Vectors
Cloning – when one gene
isn't enough...
Cloning = transfer of a DNA
fragment of interest to a selfreplicating genetic element such as
a plasmid (genomics.energy.gov)
Vector
= a molecule used as a vehicle to
carry foreign DNA into a host cell
Simplest vector = plasmid
Features of Plasmids
Size
Functions
encoded
Structure
Nomenclature
Micrograph taken by Dr.
Jack Griffith from:
http://www.accessexcell
ence.org/RC/AB/WYW/
cohen/cohen_3.php.
Why are plasmids suitable
cloning vectors?
Generally do not kill host cell
Relatively easy to purify
Can be made small
Why not use naturally
occurring plasmids?
Replication
Plasmid must contain a replicon
Replicon - an origin of replication
(oriV) plus associated cis-acting
elements
Functions of the ori
region
Host range
Narrow vs Broad
Copy number – eg - pUC vs pET
Incompatibility
Conjugation/mobilisation
Desireable Features of
Plasmid Cloning Vectors
High copy number
Small
Selectable Genes
Unique restriction sites
May have additional features such
as mob sites, RNA polymerase
promoters, etc.
pBR322
1973-1978
Bolivar and Rodriguez derivative
322
4.36 Kb; ~16 copies per cell
Narrow host range
Encodes resistance to ampicillin
and to tetracycline
How can we tell if plasmid
contains DNA of interest?
Insertional inactivation
Use BamHI site in Tetr gene for
cloning
Transform
Plate cells on?
Confirm by replica-plating on?
Early 1980s – pUC series
Features of pUC Plasmids
Small
Very high copy number
(temperature dependent)
No insertional inactivation
Blue-White Selection
Vector contains first 146 aa of the
β−galactosidase gene (lacZ)
(α−peptide)
MCS embedded within this region
Blue-White Selection
Continued
Host cell encodes carboxy terminal
portion of lacZ
Neither host nor plasmid encodes
for entire protein
Together produce enzyme that can
cleave Xgal to produce blue
precipitate
http://www.tciamerica.com/
product/bio-chem/B030.shtml
What if foreign DNA
inserted into MCS?
Foreign DNA will contain a
termination codon in the same
reading frame as the α−peptide
No α−peptide therefore no
β−galactosidase and no blue
coloured colonies
www.sigmaaldrich.com
Additional Features
Origin of DNA replication from ss
filamentous phage such as F1 or M13
Phagemid vector
Not commonly used anymore
T7 and SP6 promoters
RNA production
Sequencing primers
www.promega.com
Cloning in Plasmid Vectors
Plasmid and DNA of interest cleaved
with restriction enzyme(s) forming
compatible ends
Ligation
Cloning – Easiest method
5'
3'
GAATTC
CTTAAG
3'
5'
EcoRI cleavage creates 5'
Sticky ends
5'P
3'OH
3'OH
G
CTTAA
5'P
5'P
AATTC
G
3'OH
5'P
Inter- and Intramolecular
Reactions
vector
insert
Intermolecular reaction
vector
insert
Intramolecular reaction
Formation of a noncovalently bonded
circular molecule
Enzyme Choice
Determined by
Vector
MCS from pUC19 from Fermentas.com
Enzyme Choice
Also determined by:
Insert sequence
NEB Cutter:
http://tools.neb.com/NEBcutter2/index.
php
Cloning PCR Products
Mix PCR product and linearised vector
and carry out blunt end ligation
Will this work?
Sometimes, sort of
Problems?
Strategies
Incorporate restriction enzyme
recognition sequences into PCR
product
OR
Create specialised cloning vectors
Introduction of Restriction
Sites via PCR
Restriction site sequence
Primer
3'
5'
Template DNA
(http:www.fermentas.com/techinfo/re
/restrdigpcrii.htm)
Steps Involved
Choose restriction enzyme sequences
and build in to primers
Carry out PCR
Digest product and vector with
complementary enzymes
Ligate
Digestion of PCR Products
May be necessary to add additional
bases in order to digest linear DNA (ie,
restriction enzymes may not digest at
ends of DNA molecules)
NEB, Stratagene, Fermentas all have
online resources to consult
pGEM-T (Promega)
Why Use?
Allows H-bonds to form to stabilise
hybrid molecule
Advantages
Disadvantages
Other Options?
www.fermentas.com
Cloning 16srDNA
Through the Fermentas web site,
download instructions for CloneJET
system
Wed – plan on gel extraction, quantify
DNA via spec and gel (if time permits –
if not, finish Friday)
Determine amount of PCR product to
add for a 3:1 insert to vector molar
ratio