Gene Cloning and Plasmid Vectors

Question 1

What is gene cloning?

Answer 1

• 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

Question 2

What does gene cloning apply to?

Answer 2

• 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

Question 3

Cloning Vectors for E. coli

Answer 3

• 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

Question 4

Plasmids

Answer 4

• 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

Question 5

Introduction of Plasmid DNA into E. coli

Answer 5

• 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

Question 6

General Transformation Procedure for E. coli

Answer 6

• 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

Question 7

Production of Competent E. coli

Answer 7

• 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

Question 8

Transformation of E. coli: Heat shock

Answer 8

• 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

Question 9

How are cells ‘made’ chemically competent?

Answer 9

• 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

Question 10

Transformation of E. coli: Electroporation

Answer 10

• 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

Question 11

Origin of replication (Plasmid Cloning Vectors: Minimal Requirements)

Answer 11

• confers the ability to propagate/replicate independent from chromosomal DNA
• type of ori strongly influences number of copies/cell (copy number)

Question 12

Copu number

Answer 12

• 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.

Question 13

Advantages of high-copy number

Answer 13

• Increased yield from protein overexpression

- Cloning:Using a high-copy plasmid will generally result in greater yields from plasmid preps

Question 14

Advantages of lower copy number

Answer 14

• 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

Question 15

Selectable marker as minimal requirement for plasmid cloning vectors

Answer 15

• 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

Question 16

Multiple cloning site (MCS) as minimal requirement for plasmid cloning vectors

Answer 16

• 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

Question 17

Plasmid Cloning Vectors: Smaller is Better

Answer 17

• 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

Question 18

pBR322

Answer 18

• 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)

Question 19

pUC Series Vectors

Answer 19

• 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

Question 20

pZErO-2

Answer 20

• Contains the lethal E. coli gene, ccdB
• Acts by poisoning bacterial DNA-gyrase (topoisomerase II)
  Prevents the unwinding of DNA

Question 21

Testing for Insert: Colony PCR

Answer 21

• 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

Question 22

Host – Vector Choice

Answer 22

• 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

Question 23

Using PCR to Introduce Cloning Handles

Answer 23

• 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

Question 24

General cloning strategy

Answer 24

• 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)

Question 25

Gibson Assembly - Principle

Answer 25

- dsDNA fragments with overlapping ends - Gibson Assembly Master Mix (5’-exonuclease, DNA polymerase, DNA ligase) - 50oC for 1 hour - Fully assembled DNA fragment

Question 26

Gibson Assembly - Mechanism

Answer 26

- 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

Question 27

Site-directed mutagenesis using Gibson Assembly

Answer 27

- 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

Question 28

Requirement for Cloning Procedures: Pure DNA

Answer 28

- 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

Question 29

DNA Purification for Cloning: Size/Type of Molecule

Answer 29

- 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

Question 30

Plasmid purification from bacterial cell culture

Answer 30

- Generally known as ‘MiniPrep’, can be purchased as a complete kit - Many suppliers using similar protocols - Selectively purifies plasmid DNA from genomic DNA

Question 31

Protocol/method of plasmid purification

Answer 31

- 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)

Question 32

PCR/Restriction Digest/Ligase DNA purification

Answer 32

- Generally known as ‘PCR purification kit’ or ‘DNA clean up kit’ - Many suppliers using similar protocols

Question 33

Protocol/method of PCR/Restriction Digest/Ligase DNA purification

Answer 33

- 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)

Question 34

Protocol/method of gel extraction kit

Answer 34

- 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)

Question 35

UV/VIS analysis

Answer 35

- 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

Question 36

Gel electrophoresis

Answer 36

- 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