Gene Cloning and Plasmid Vectors

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

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

• 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.

• Increased yield from protein overexpression - Cloning:Using a high-copy plasmid will generally result in greater yields from plasmid preps

• 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

• 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

• 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

• 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

• 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)

• 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

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

• 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

• 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

• 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

• 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)