15 March 2016 Today’s Title: CW: Introduction to genetic engineering Learning Question: what is genetic engineering?

Aims from specification (i) explain how isolated DNA fragments can be placed in plasmids, with reference to the role of ligase; (j) state other vectors into which fragments of DNA may be incorporated; (k) explain how plasmids may be taken up by bacterial cells in order to produce a transgenic microorganism that can express a desired gene product;

Key words Genetic engineering Restriction enzymes Sticky ends Annealing Restriction fragments DNA ligase Recombinant DNA Vector

What is genetic engineering? Organisms that have their DNA altered by genetic engineering are called transformed organisms Transformed organisms have recombinant DNA that is formed by joining together DNA from different sources Usually extraction of a gene from one organism and inserting it into another organism (usually from a different species) Genes can also be manufactured, instead of extracting them from organisms The organism that contains the desired gene produces the desired protein product e.g. insulin An organism that has been genetically engineered to include a gene from a different species is sometimes called a transgenic organism

Basic steps of genetic engineering 1.The required gene is obtained 2.A copy of the gene is placed (packaged and stabilised) in a vector 3.The vector carries the gene to the recipient cell 4.The recipient expresses the gene through protein synthesis A variety of approaches may be used at each stage. More detail on these later.

The role of restriction and ligase enzymes Genetic engineering – replace, modify or add DNA coding sequences to a living organism. Uses enzymes to “cut” up parts of DNA from one organism and “stick” them into another. Resulting new organism will incorporate genes and therefore proteins from imported DNA.

The role of restriction and ligase enzymes Describe the process taking place in the diagrams opposite 1.Isolating and identifying gene> protein 2.Putting isolated gene into another organism using a vector 3.Cloning the organism to make several copies of the imported gene product.

The role of restriction and ligase enzymes Restriction enzymes (restriction endonucleases) cut DNA at particular sequences of bases. Two types: – Enzymes that cut straight across – blunt ends – Enzymes that cut in a staggered way – sticky ends (more useful – need to know this)

Sticky ends Short stretches of single stranded DNA are complementary to each other. If both ends are cut with the same enzyme, the sticky ends will stick together by complementary base paring, forming hydrogen bonds Annealing is the name given to the process by which sticky ends form hydrogen bonds.

Restriction fragments DNA has several recognition sites where restriction enzymes can “cut” at very specific points. These restriction sites are only 4-8 base pairs long, so they are specific sites. Cuts at restriction sites produces lots of DNA fragments of different lengths. The resulting fragments of DNA are known as restriction fragments.

Bacteria and DNA Restriction enzymes are produced naturally by bacteria as a defence mechanism against viruses. The restrict viral growth Restriction enzymes used in GE are named after the bacteria it came from, e.g. EcoR1 – e.coli strain R was the first to be identified.

Cutting and pasting Once DNA fragments have been annealed to DNA of another organism, the broken DNA needs to be repaired. Remember annealing is the formation of complementary base pairs – this is done by weak hydrogen bonds. DNA ligase enzymes repair broken DNA by forming covalent bonds between the new sections (condensation reaction.

restriction endonuclease enzymes ligase enzymes Describe the stages of genetic engineering in this diagram

Recombinant DNA Technology Bacterial cell with a __________ selected Plasmid removed and cut Open using ________ __________. Desired Gene cut out of Chromosome using __________ _________ The desired gene is _________ in human Gene sealed in Plasmid using ____. Which joins ______ ends together. isolatedplasmid restriction endonuclease restriction endonuclease ligase plasmid

Recombinant DNA Technology Plasmid inserted into ________ _______ _______ ______ _______ Used to check the gene is present Recombinant bacteria Allowed to ________ rapidly Bacteria ________ that _______ the gene as a protein which is then _________. hostbacterium cell DNA probe multiply identified propagated expresses Human proteins produced by this method include _____ (trade name humulin), _____ ______ ______ (HGH)

Marker genes Only a small percentage of cells take up the recombinant DNA. Identifying these cells is important. This can be done with marker genes. Marker genes, when expressed, display easily observable characteristics. When recombinant DNA is transferred into a vector of the host cell, the marker gene goes with it. This means that if we can see the expression of MG we know that GE has been succesful.

Antibiotic resistance markers and reporter systems Marker genes can code for resistance to particular antibiotics. GE organisms grown in a growth medium containing antibiotics will do this. Only GE organisms will grown in this environment as the antibiotic will kill other organisms that fail to take up the plasmid. This technique has worked well, but there is concern about spreading “resistant genes”.

Antibiotic resistance markers and reporter systems Reporter systems have superseded antibiotic resistant genes. This involves bioluminescence – target genes will fluoresce, allowing them to be followed/isolated

Another route to isolating a gene protein coding genes in the human genome. Isolating specific genes is difficult – complementary DNA technique is used Complementary DNA (cDNA) is copied from mRNA The enzyme reverse transcriptase synthesises DNA from an RNA template. Certain retroviruses have this enzyme capable of doing this. DNA produced is single stranded, but DNA polymerase can be used to produce a double stranded cDNA “gene”. Beta cells in the pancreas make the hormone insulin, therefore manufactures mRNA that codes for insulin. mRNA can be isolated and used to make cDNA for the human insulin gene.

Another route to isolating a gene Advantages of GE using bacteria – Microorganisms are simple and easy to use – Quick and easy to culture – Few ethical issues about their use Disadvantages of GE using bacteria – Cannot make human proteins – Animals can be used, but products have to be extracted via milk/urine and not directly from cells – Plants can be used – products secreted via roots.