PEF - Molecular Cloning

Molecular cloning is the first laboratory step in producing a recombinant protein. The aim is to obtain a plasmid that carries the gene of interest (GOI) in an expression vector.

A general methodology is described briefly below:

1. Obtain/generate a DNA template of the GOI
2. Ligate the GOI into an appropriate expression vector (ligation dependent cloning)
3. Transform the expression vector into a bacterial strain
4. Analyse bacterial clones to confirm integration of the GOI
5. Sequence verification

Cloning Strategies

There are a variety of cloning strategies available and they can be broadly classified into 2 categories as shown in the diagram below.

Useful articles on several of these cloning strategies can be found on the related articles page.

Site-directed Mutagenesis (SDM)

SDM is a molecular technique used to introduce specific mutations (deletions/insertions/point mutation) to the GOI or vector sequence. SDM is commonly used for mutations involving:

• Restriction endonuclease recognition sites
• Start or stop codons
• Fusion tags and signal peptides
• Optimisation of transcriptional elements on the vector

Codon Optimisation

Codon optimisation is a tool that can be used to improve expression levels of heterologous recombinant proteins. The process involves modifying codons within a gene sequence to match the codon frequency of the intended expression host, theoretically enabling higher production rates.