Restriction cloning of a gene into a plasmid

Molecular cloning is the joining of a chosen piece of DNA to a plasmid vector so that bacteria can copy it faithfully, millions of times over, each colony a pure clone. The approach was established when restriction-enzyme fragments of two plasmids were joined in vitro and put into Escherichia coli, where the new, recombinant plasmid replicated and passed on the properties of both parents (Cohen et al., 1973). The same principle applies here: the vector and the insert are cut so that their ends match, joined, and carried by a single bacterium.

The pieces are prepared with three kinds of enzyme. Restriction endonucleases cut double-stranded DNA at specific short sequences, usually leaving short single-stranded overhangs ("sticky ends") that pair only with a matching cut; using two different enzymes gives the insert two different ends, so it can only drop into the vector one way round (directional cloning). A DNA ligase then seals the breaks. The insert itself is usually copied first by the polymerase chain reaction with a high-fidelity, heat-stable polymerase (Saiki et al., 1988), using primers that carry the restriction sites, plus a few extra bases, as tails.

The joined plasmid is put into competent E. coli, cells treated so they take up DNA. The method rests on the finding that bacteria made permeable with cold calcium chloride can be infected by, and transformed with, DNA (Mandel & Higa, 1970), later refined into the high-efficiency transformations used today (Hanahan, 1983). Each cell that takes up a plasmid, and only such a cell, grows into a colony on a plate containing the antibiotic whose resistance gene the plasmid carries. Where the vector is a general cloning plasmid that carries the lacZα fragment, an interrupted insert site turns the usual blue colonies white, a quick first screen (Yanisch-Perron et al., 1985).

Not every colony carries the intended construct, so colonies are screened, by colony PCR (Güssow & Clackson, 1989) or a diagnostic restriction digest, and the final proof is to read the sequence across the junctions and the insert (Sanger et al., 1977). A verified clone is kept as a frozen glycerol stock. The gene cloned here is a harmless fluorescent-protein gene, the kind first used as a marker of gene expression (Chalfie et al., 1994); every sequence, plasmid and strain here is simulated for teaching and does not correspond to a real one. An alternative, sequence-independent route, Gibson assembly, joins fragments through overlapping ends in a single reaction without restriction sites (Gibson et al., 2009).

Learning objectives

• Read a plasmid map and name the parts a cloning vector needs: a promoter, a multiple cloning site, an antibiotic-resistance gene and an origin of replication
• Choose a pair of restriction enzymes from the multiple cloning site for directional cloning, avoiding any enzyme that also cuts inside the insert
• Design PCR primers that add the restriction sites, with extra bases beyond them so the enzymes can cut, and amplify the insert
• Decide whether two enzymes can be used in one buffer, digest the insert and the vector, and dephosphorylate the vector to stop it re-closing on itself
• Gel-purify the cut DNA, and work out the insert-to-vector molar ratio from nanograms and base pairs before ligating with T4 DNA ligase
• Transform E. coli, set up vector-only, vector-plus-ligase and uncut-vector controls, screen colonies by colony PCR or diagnostic digest, and confirm the clone by sequencing before freezing a glycerol stock

Ethics and safety

This work makes a genetically modified organism (E. coli carrying an engineered plasmid) and in a real laboratory requires prior approval by an Institutional Biosafety Committee (in India, under the Rules, 1989 of the Environment (Protection) Act 1986, with comparable national rules elsewhere) and must follow the containment and waste-disposal rules for the host strain. It is kept strictly to standard, non-pathogenic laboratory strains of E. coli (K-12 type) used for cloning and to a harmless insert, a fluorescent-protein gene; no pathogen or toxin genes are used, and no work is done that would make an organism more harmful. General laboratory safety training is needed: DNA stains and the ultraviolet or blue light used to view gels are hazards, so gloves and eye or face protection are worn, and all reagents and transformed cultures are handled and disposed of as the local risk assessment and genetic-modification regulations require. Constructs and their full sequences are recorded so the work can be checked and repeated.

Key terms

• Plasmid vector: A small circular DNA molecule that replicates in bacteria independently of the chromosome and is used to carry a cloned piece of DNA. A cloning vector carries the parts needed to select and copy it.
• Insert: The piece of DNA being cloned: here a gene for a fluorescent protein, amplified by PCR and cut so its ends match the vector.
• Multiple cloning site: A short stretch of the vector containing many unique restriction-enzyme sites close together, into which the insert is placed. Often abbreviated MCS, or called a polylinker.
• Restriction enzyme: A bacterial enzyme (a restriction endonuclease) that cuts double-stranded DNA wherever a specific short sequence occurs, often leaving a single-stranded overhang.
• Sticky end: A short single-stranded overhang left by many restriction enzymes. Two pieces cut by the same enzyme have complementary overhangs and pair up, which is how the insert and vector join.
• Directional cloning: Cutting the insert and vector with two different enzymes so each end is different. The insert can then join the vector in only one orientation, and the vector cannot simply re-close on itself.
• Origin of replication: The sequence in the plasmid where its copying begins; it lets the plasmid replicate inside the bacterium and sets how many copies each cell holds. Often called the ori.
• Antibiotic-resistance gene: A gene on the plasmid (here for resistance to ampicillin) that lets only cells carrying the plasmid grow on a plate containing that antibiotic; it is the selectable marker.
• Dephosphorylation: Removing the 5' phosphate groups from the cut vector with a phosphatase. Without a phosphate the vector's two ends cannot be sealed to each other, so it cannot re-close without an insert.
• DNA ligase: An enzyme (here T4 DNA ligase) that seals the breaks between the insert and the vector, forming the continuous circular recombinant plasmid.
• Molar ratio: The ratio of the number of molecules of insert to vector in the ligation, not their masses. Because the insert is shorter than the vector, equal masses are not equal numbers, so the ratio is worked out from nanograms and base pairs.
• Competent cells: Bacteria treated (here with cold calcium chloride) so that their surface will take up DNA. A brief heat shock then drives the plasmid in.
• Blue-white screening: A first screen available with vectors carrying the lacZα fragment: an empty vector gives a working enzyme and blue colonies on X-gal, while an insert interrupts the fragment and gives white colonies. It does not replace sequencing.
• Glycerol stock: A culture of a verified clone mixed with glycerol and frozen at −80 °C, from which the strain can be revived indefinitely.