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Gene cloning + plasmid vectors, Compare heat shock and electroporation,…
Gene cloning + plasmid vectors
DNA purification
Why?
Purification of a DNA fragment
Removal of chemicals/contaminant enzymes etc
PCR products
Ligation reactions
Purity
How?
Plasmid purification from bacterial cell culture
'Miniprep'
Bought as complete kit
Protocol
Grow cell culture containing bacteria propagating plasmid
Harvest cells by centrifugation
Lyse cells with detergent
Add RNAase to remove RNA
Neutralise
1 more item...
PCR purification kit/DNA clean-up kit
Protocol
Dilute reaction mixture in DNA binding buffer
DNA binds
Wash to remove buffer components, enzymes + small DNA fragments
Elute into ultrapure water or slightly basic (pH 8.5) low strength buffer
Gel extraction kit
Protocol
Excise DNA fragment from agarose gel
Mix gel slice with gel dissolving buffer + heat to dissolve agarose
Apply to column - DNA binds
Wash to remove buffer + residual agarose
Elute into ultrapure water or slightly basic (pH 8.5) low strength buffer
DNA analysis
UV/VIS
Increases absorbance at \(\lambda\) = 230nm
Caused by proteins/other contaminants
Ratio between A\(_{260}\)/A\(_{230}\) and A\(_{260}\)/A\(_{280}\)
Should be between 1.8-2.0
Indicators of DNA purity
Proteins absorb at maximum \(\lambda\) = 280nm
Nucleic acids absorb at maximam \(\lambda\) = 260nm
Conversion factor for [DNA]
A\(_{260}\) = 1
Corresponds to 50ng\(\mu\)L\(^{-1}\)
If path length = 1cm
Does not inform on homogeneity of different sized fragments
i.e. cannot distinguish DNA fragments
Gel electrophoresis
Quantification of bands
Based on markers
Quality control of samples expected to contain fragments
Destructive analysis
Sample cannot be used aftor
Gene cloning
In vivo method to amplify DNA fragments
Large (\(\mu\)g) quantities
Facilitates modification of DNA molecules
Applications
Generation of DNA probes
Detection of genes/mutations
Production of recombinant proteins
DNA sequencing
Investigating genetically engineered sequences
Vector
Entity that transports something
E.coli
Widely used organism for cloning
Many DNA delivery vectors available
Based on 2 naturally occurring replicons
Plasmids
Bacteriophages
Plasmids
dsDNA
Circular
Supercoiled
Naturally occurring plasmids
Genetically mobile
Genes encode non-essential function
Can propagate in more than one bacterial strain
i.e. enable horizontal gene transfer
Allow e.g. spread of antibiotic resistance to spread amongst species
Artificial/engineered plasmids
Typically specific to
E.coli
Carry specific marker
e.g. antibiotic resistance
Capable of autonomous replication
Multiple copies in one cell
Contain origin of replication
Introduction of recombinant DNA to host
Transfection or transformation
Cell/organism that can be transformed is competent
Natural competence exists
e.g.
Bacillus sp
E.coli
is not naturally competent
Can be 'forced'
Harvest cells in exponential growth phase
Cool on ice immediately after log phase
Growth at lower temp (20-25\(^o\)C)
Due to altered membrane composition
Competent cells can be stored at -80\(^o\)C if immediately flash frozen (liquid N\(_2\)) after production
Main methods for transformation
Heat shock
Cells made chemically competent
Divalent metal ions
Ca\(^{2+}\)
Mg\(^{2+}\)
Mn\(^{2+}\)
Polyethylene glycol
Dimethyl sulfoxide
All thought to concentrate DNA on cell surface or make cell membrane more porous
early methods used CaCl\(_2\)
Transformation triggered by incubating for short periods (30-60s) at 42\(^o\)C
Increased temp. makes membrane more permeable/lower membrane potential
Possible entry via Bayer's junction on inner/outer membrane
Electroporation
Discharge of high voltage over short distance
Prep. of electrocompetent
E.coli
Wash in ultrapore water containing 10%(v/v) glycerol
Glycerol is cryoprotectant
Repeated washing removes salts
More efficient than heat shock
Both rely on temporarily making
E.coli
cell membrane porous to plasmid DNA
Recovery needed after transformation
In rich growth medium
Most efficient procedures
Only 1 in 20 plasmid DNA successfully transformed
Plasmic cloning vectors
Minimal requirements
Origin of replication (ori)
Confers ability to propagate independent from chromosomal DNA
Type of ori strongly influences no. copies produced
Low medium or high
Selectable marker
Coding for phenotypic traits e.g. antibiotic resistance
Transformats distinguishable from non-transformats
Non-transformed can't grow
Multiple cloning site (MCS)
Single region for large no. of different, unique RE recognition sites
RE digestion results in linearisation rather than fragmentation
Enables restriction fragments produced by numerous RE to be cloned
Smaller is better
Transformation efficiency decreases with increasing plasmid size
Reduction in no. restriction sites outside intended ligation site
Lower metabolic burden
Typically higher copy no.
Easier to purify
Less prone to fragmentation due to shearing
pBR322
Relatively small - 4361bp
Low copy no. ~15
2 antibiotic resistance genes
Amp\(^R\) + Tc\(^R\)
Transformats + non-transformats grow on Amp plate
Use wooden touch block to transfer colonies onto Tet plate
Transformats sensitive to Tet
Go back to Amp plate + select recombinant colonies based on comparing position of growth/non-growth on Tet plate
pUC series vectors
Derived from pBR322
Only Amp\(^R\) and
ori
remain
Proper MCS
Located in LacZ\(\alpha\) fragment
Complementary to \(\beta\)-galactosidase
Blue/white screening
Active \(\beta\)-galactosidase converts X-gal into 5-bromo-4-chloro-3-hydroxy-indole
Indigo under aerobic conditions
If fragment is successfully inserted into MCS
LacZ\(\alpha\) reading frame disrupted
No active \(\beta\)-galactosidase
Transformat colonies are white
Non-transformat colonies are blue
High copy no. ~500-700
pZErO-2
Selective marker
Neomycin/Kanamycin/Geneticin resistance
Contains lethal
E.coli
gene, ccdB
Insertion of fragment disrupts gene and cell survives
Testing for insert
Colony PCR
Marker to screen/select is not always possible
Flanking primers
Many plasmids have binding sites for sequencing primers on either side of MCS
Used as primer pair for PCR
Host-vector choice
Ease of cloning into MCS
Propagating in the optimal host
Stability of DNA produced after purification
E.coli
genotype
endA1
does not have endonuclease 1 activity
Lower risk of DNA degradation in storage
Correct machinery for protein expression
Compare heat shock and electroporation
Advantages/disadvantages of high copy numbers