Transformation and Cloning

1. Transformation and Cloning

2. Transformation • Remember that a gene is a piece of DNA. • It provides the instructions (codes) for a protein that gives an organism a particular trait. Genetic transformation • This is the uptake of naked DNA from the environment. • Can induce this to happen in a laboratory situation – inserting the DNA you want the microbe to express. • Uptake of new DNA by microorganism will provide cell with new characteristics. • This technique used widely in biotechnology.

3. In the laboratory situation • From a population of 1000 or more treated cells only one or two may pick up foreign DNA. • Those which do are said to be transformed.

4. Bacterialtransformation Antibiotic-sensitive bacterial cell Calcium chloride treatment permeablises cell wall Plasmid DNA Transformed bacterial cell Transformed bacteria selected by growing on solid media containing appropriate antibiotic

5. Recipients for foreign DNA • Bacterium E. coli and the yeast S. cerivisiae are widely used as recipients for foreign DNA. • Both are unicellular and quick growing. • Ideal for large-scale production of proteins such as industrial enzymes. Discuss the advantages and disadvantages of using E. coli and S. cerivisiae.

6. E. coli advantages • Several different types of plasmid can be used to introduce foreign DNA. • Foreign DNA can account for up to 60% of its total protein production. • Fast growing. • Easy to transform. • Easy to manipulate.

7. E. coli disadvantages • Does not carry out post-translational modifications to proteins. • These modifications are necessary for protein function in, for example, human cells. • Bacterium occurs naturally in the intestines of humans and under certain circumstances can cause disease.

8. S. cerivisiae advantages • Rarely a human pathogen. • Genes organised, expressed and controlled in ways that are similar to human genes. • Carries out post-translational modifications, eg addition of sugar residues, which are a common feature of human proteins.

9. S. cerivisiae disadvantages • Difficult to transform. • Yields less protein than bacteria. • Plasmids easily lost from yeast – no selective advantage to yeast.

10. Cloning vectors

11. Cloning vectors • In gene cloning, once recombinant DNA (rDNA) has been constructed it is introduced into a host. • In the host, rDNA has to be: maintained replicated passed from one generation to another. • This is achieved by introducing rDNA into a cell on a DNA vehicle called a cloning vector – most commonly used are plasmid cloning vectors.

12. Plasmids • Extra-chromosomal DNA found in bacteria. • Loops of double-stranded DNA. • Some present in multiple copies. • Independently replicate inside bacteria. plasmid chromosome

13. Artificial plasmids • Artificial plasmids have been genetically engineered for the purpose of cloning. • Used as vectors, ie to transfer DNA from one cell to another.

14. Plasmids • Purpose of a vector is to carry DNA into a cell. • Foreign DNA must be cut with same enzyme as plasmid so the ‘ends’ are compatible – plasmid and foreign DNA are then joined by ligase enzyme.

15. Features of plasmids Plasmids also contain a selectable marker. • Usually an antibiotic resistance gene: • required for maintenance of plasmid in the cell • advantageous for bacteria to keep the plasmid (can then grow in presence of antibiotic). • Commonly used selectable markers are ampicillin, neomycin and chloramphenicol.

16. Example of controlling expression using an inducer Gene for green fluorescent protein from jellyfish can be inserted into various other cell types. Gene has been combined in a plasmid along with control elements from arabinose operon ( ie usually regulates synthesis of enzymes needed to metabolise the sugar arabinose). Can turn expression of the ‘glowing protein’ on by adding arabinose to the growth medium.

17. Alba the pGLO bunny!

18. Steps in transforming bacteria Bacteria incubated with plasmid (with appropriate promoter and antibiotic resistance gene). Bacteria plated out onto agar plates containing antibiotic. Only those bacteria that have taken up plasmid will be able to grow on agar plus antibiotic. Transformed bacteria can then be isolated and grown in bulk with appropriate antibiotic. Bacteria multiply to produce genetically identical offspring – clones.