Gene Cloning and Plasmid Vectors Flashcards
1
Q
What is gene cloning?
A
- In vivo method to amplify DNA fragments, creating copies of a specific DNA sequence in large (µg-scale) quantities
- In addition to producing specific DNA molecules/fragments in large quantities it also facilitates modification of these DNA molecules
2
Q
What does gene cloning apply to?
A
- Generation of DNA probes
- Detection of presence/absence of particular genes or mutations
- Production of recombinant proteins (expression cloning)
- DNA sequencing (Sanger method)
- Investigate genetically engineered sequences
3
Q
Cloning Vectors for E. coli
A
- Vector (an entity that transports something): plasmid vectors can carry recombinant genetic material into a cell
- E.coli is one of the most widely used organisms used for cloning: wide variety of DNA delivery vectors are available for the replication of DNA in E.coli
- They are based on 2 naturally occurring replicons: plasmids, bacteriophages
4
Q
Plasmids
A
- Double-stranded circular supercoiled DNA molecules
- Naturally occurring plasmids are often genetically mobile: genes typically encode non-essential functions, can propagate in more than one bacterial strain, i.e. they enable horizontal gene transfer, allow e.g. antibiotic resistance to spread among multiple species
- Artificial/engineered plasmids used in molecular cloning are typically specific to E. coli and carry a selective marker such as antibiotic resistance
- Capable of autonomous replication: multiple copies/E. coli cell, contain an origin of replication
5
Q
Introduction of Plasmid DNA into E. coli
A
- Introduction of recombinant DNA into a host: transfection or transformation
- A cell/organism that can be transformed is competent
- Natural competence exists; disadvantage: naturally competent organisms are genetically unstable, undesirable for cloning
- E. coli isn’t naturally competent, but can be transformed ‘by force’
- Main methods of transformation for E. coli: heat shock, electroporation
- Both methods rely on temporarily making E.coli cell membrane more porous, allow plasmid DNA to enter the cell
6
Q
General Transformation Procedure for E. coli
A
- heat shock or electroporation
- recover: methods of transformation are stressful to cell, need for recovery in rich growth medium
- transfer to selective growth medium (agar plates)
- overnight growth
7
Q
Production of Competent E. coli
A
- Grow E. coli cells in liquid culture to mid-exponential growth phase: growth at lower temps (20-25oC instead of 37oC) is thought to result in higher competency, most likely due to altered membrane composition
- Cool cells by placing the culture flask on ice prior to ‘competency enhancing procedure’
- Competent cells can stored for several months at -80oC, provided they were immediately flash frozen (liquid N2) after production
8
Q
Transformation of E. coli: Heat shock
A
- Actual transformation is triggered by incubating for a short period (30-60s) at 42oC
- Increased temperature is expected to temporarily make the membrane more permeable/lower membrane potential
- Possibly entry via Bayer’s junction on inner/outer membrane
9
Q
How are cells ‘made’ chemically competent?
A
- by treatment with several chemicals or combinations thereof: divalent metal ions (e.g. Ca2+, Mg2+, Mn2+), polyethylene glycol (PEG), dimethyl sulfoxide (DMSO)
- All ingredients are thought to either concentrate DNA on the cell surface (divalent metal ions, PEG) or make the cell membrane more porous (DMSO, divalent metal ions)
- Early methods mainly used CaCl2: CaCl2-competent cells has become almost synonymous with chemically competent cells/heat shock treatment
10
Q
Transformation of E. coli: Electroporation
A
- Method: discharge of high voltage over a short distance temporarily causes the formation of membrane pores
- Preparation of electrocompetent E. coli involves repeated washing of cells with ultrapure H2O containing 10% (v/v) glycerol
- Repeated washing done to remove salts
- Preparation procedure has no direct influence on competency, electroshock is what causes the competency
11
Q
Origin of replication (Plasmid Cloning Vectors: Minimal Requirements)
A
- confers the ability to propagate/replicate independent from chromosomal DNA
- type of ori strongly influences number of copies/cell (copy number)
12
Q
Copu number
A
- the average or expected number of copies per host cell
- Plasmids are either low, medium or high copy number
copy number depends on three main factors: oriand its constituents – ColE1 RNA I and RNA II; size of plasmid and its associated insert, larger inserts and plasmids may be replicated at a lower number as they represent a greater metabolic burden for the cell; culture conditions: factors that influence the metabolic burden on the host.
13
Q
Advantages of high-copy number
A
- Increased yield from protein overexpression
- Cloning:Using a high-copy plasmid will generally result in greater yields from plasmid preps
14
Q
Advantages of lower copy number
A
- Expression of ‘toxic’ protein product: a low-copy might be better to minimise toxic effects/energetic burden from protein expression
- Expression to simulate ‘natural’ conditions for in vivo experiments: artificially high protein expression/RNA transcription may give rise to artifacts
15
Q
Selectable marker as minimal requirement for plasmid cloning vectors
A
- Coding for phenotypic traits e.g. antibiotic resistance (AmpR) or colour change in presence of medium components
- Transformants must be distinguishable from non-transformants
- Non-transformed bacteria have no vector (plasmid or phage) so cannot grow on media with antibiotic
16
Q
Multiple cloning site (MCS) as minimal requirement for plasmid cloning vectors
A
- Single region for large number of different, preferably unique restriction endonuclease recognition sites
- Restriction enzyme digestion results in linearisation of plasmid vector rather than fragmentation into several fragments
- Enables restriction fragments produced by numerous restriction enzymes to be cloned
17
Q
Plasmid Cloning Vectors: Smaller is Better
A
- Transformation efficiency decreases with increasing plasmid size
- Reduction in number of restriction sites outside the intended ligation site
- Lower metabolic burden for plasmid propagation, i.e. replication of DNA costs energy
- Typically higher copy number
- Easier to purify
- Less prone to fragmentation due to shearing
18
Q
pBR322
A
- One of the first vectors to be developed
- comparatively small in size - 4361bp
- Reasonably low copy number ~15
- contains 2 Antibiotic resistance genes (AmpR + TcR): each resistance gene contains unique restriction sites allowing non-recombinants to be distinguished from recombinants (insertion of DNA fragments in one of the respective resistance genes will abolish antibiotic resistance)
19
Q
pUC Series Vectors
A
- Derived from pBR322 although only the AmpR gene and ori remain
- Contains a proper multiple cloning site
- High copy number (500-700)
- Multiple cloning site located in the LacZa fragment which complements b-galactosidase
blue/white screening
20
Q
pZErO-2
A
- Contains the lethal E. coli gene, ccdB
- Acts by poisoning bacterial DNA-gyrase (topoisomerase II)
Prevents the unwinding of DNA
21
Q
Testing for Insert: Colony PCR
A
- Marker to screen/select for successful fragment insertion not always possible (typically absent in plasmids designed for protein overexpression)
- Colony PCR with flanking primers can be used: many plasmids have binding sites for sequencing primers on either side of MCS that can be used as primer pair for a PCR reaction
22
Q
Host – Vector Choice
A
- Ease of cloning into MCS: are optimal sites for digesting your insert compatible with the MCS etc.
- Propagating in the correct/optimal host (copy number, ori): expression in non-standard bacteria may be facilitated by use of shuttle vectors, i.e. vectors that can propagate in e.g. E. coli and the non-standard bacterium to take advantage of the easy of DNA manipulation in E. coli
- Stability of produced DNA after purification: E.coli genotype endA1 doesn’t have Endonuclease I activity, less risk of DNA degradation while stored
- Correct machinery for protein expression
23
Q
Using PCR to Introduce Cloning Handles
A
- PCR can be used to introduce extra bases via extensions beyond target DNA sequence introduced at the 5’-end of both forward (F) and reverse (R) primers
- This extra bit of sequence can be used to introduce cloning handles such as restriction sites or overlapping regions for Gibson Assembly
24
Q
General cloning strategy
A
- DNA clean-up or gel extraction to remove restriction enzymes and unwanted fragments from plasmid
- target gene added
- joined by DNA ligase
- forms recombinant plasmid
- Transformation
(DNA clean-up prior to transformation optional)
25
Gibson Assembly - Principle
- dsDNA fragments with overlapping ends
- Gibson Assembly Master Mix (5’-exonuclease, DNA polymerase, DNA ligase)
- 50oC for 1 hour
- Fully assembled DNA fragment
26
Gibson Assembly - Mechanism
- 5’-exonuclease digests only a single strand from the 5’-end
- DNA fragments anneal
- DNA polymerase extends 3’ ends
- DNA ligase seals nicks
- Overlapping ends can be generated by including overlapping extensions in e.g. PCR primers
27
Site-directed mutagenesis using Gibson Assembly
- Perform two separate PCR reactions (A and B) to introduce a mutation into a fixed position in the gene using a sense (mS) and anti-sense (mAS) mutagenic primer
- Combine PCR products A and B with relevant Gibson assembly compatible vector all in a single reaction
28
Requirement for Cloning Procedures: Pure DNA
- DNA needs to be ‘pure’ in order to be able to manipulate in vitro
- Preparative restriction digest of PCR products: high salt, exonuclease activity of the thermostable DNA polymerase
- Transformation of Ligation reactions: high salt can interfere with effective transformation to E. coli, in particular electroporation
- Purity: interference of contaminants during the measurement of DNA concentration by UV/VIS spectroscopy prevents accurate determination
- In particular digests and some PCR reactions can result in multiple DNA molecules, only one of which is desired and the other fragments may interfere with follow-up reaction
29
DNA Purification for Cloning: Size/Type of Molecule
- Purification of a DNA fragment of a particular size from other nucleic acids
- MiniPrep: Selectively purifies plasmid DNA and removes RNA and chromosomal DNA
- Separation of DNA fragments of a particular size using agarose gel electrophoresis followed by subsequent excision of the desired fragment from the agarose gel
30
Plasmid purification from bacterial cell culture
- Generally known as ‘MiniPrep’, can be purchased as a complete kit
- Many suppliers using similar protocols
- Selectively purifies plasmid DNA from genomic DNA
31
Protocol/method of plasmid purification
- Grow cell culture with bacteria propagating plasmid DNA overnight and harvest cells by centrifugation
- Lyse the cells (detergent) + removal of RNA (RNAse added)
- Neutralize (Proteins precipitate) and centrifuge
- Apply supernatant to column, only plasmid DNA binds
- Wash to remove contaminants
- Elute into Ultrapure H2O or slightly basic (pH 8.5) very low strength buffer (e.g. 1 mM Tris)
32
PCR/Restriction Digest/Ligase DNA purification
- Generally known as ‘PCR purification kit’ or ‘DNA clean up kit’
- Many suppliers using similar protocols
33
Protocol/method of PCR/Restriction Digest/Ligase DNA purification
- Dilute reaction mixture (PCR, DNA digest etc..) in DNA binding buffer and apply to column, DNA binds
- Wash to remove buffer components, enzymes (DNA polymerases, Restriction endonucleass, Ligase) and small DNA fragments (Primers, small fragments that occur after digests etc..)
- Elute into Ultrapure H2O or slightly basic (pH 8.5) very low strength buffer (e.g. 1 mM Tris)
34
Protocol/method of gel extraction kit
- Excise DNA fragment from agarose gel (if high % (>2% w/v agarose) so-called ‘low melting point’ agarose is used)
- Mix gel slice with gel dissolving buffer and heat to dissolve agarose
- Apply mix to column, DNA binds
- Wash to remove buffer components and residual agarose
- Elute into Ultrapure H2O or slightly basic (pH 8.5) very low strength buffer (e.g. 1 mM Tris)
- Many companies sell the dissolving buffer as an add-on to their regular DNA clean-up kit (i.e. columns are identical)
35
UV/VIS analysis
- Nucleic acids have an absorbance maximum at 260 nm, whereas protein contaminants have their maximum at 280 nm
- Increased Absorbance at 230 nm can be caused by proteins and/or other contaminants
- Ratio between A260/A230 and A260/A280 used as indicators for DNA purity and should be in between 1.8-2.0
- Conversion factor A260 to DNA concentration: A260 = 1 corresponds to [dsDNA] = 50 ng mL-1 (if pathlength = 1 cm)
- This analysis does not inform on the homogeneity of the size of the DNA fragments
36
Gel electrophoresis
- Separation of nucleic acids based on differences in size and shape
- Quantification of individual bands is possible (based on the amounts given for the marker), allowing for estimates as to which fragments are dominant in a mixture
- Quality control of samples expected to contain a DNA fragment/molecule of a particular length
- Destructive as an analytical method: the sample is lost after the analysis
