Genetic Engineering

Key Words

Recombinant DNA- the genetic material from 2 species that has been combined Transgenic Organism- an organism with DNA from another species introduced Donor DNA- the DNA that is introduced Host- the organism that has the DNA introduced Transformed Cell- incorporated plasmid with a foreign gene Restriction Endonuclease- bacterial enzymes that cut DNA at a specific nucleotide sequence Sticky End- a sequence of unpaired bases on a double stranded DNA that readily base airs with a complementary strand Reverse Transcriptase- an enzyme derived from a retrovirus that catalyses the synthesis of cDNA from an RNA template

Isolating the Gene

Restriction endonucleases are used to cut the DNA into many small fragments, to isolate specific genes. They usually leave a staggered cut, leaving a sticky end.Example of restriction endonuclease: EcoR1 from E.coli, this breaks DNA where guanine is next to adenine. Leaves a sticky end. The sticky ends on each strand are in reverse order of each other (palindrome)Drawbacks of using endonuclease: If the base sequence that the enzyme cuts is in the gene that needs to be isolated, then the gene will be broke into fragments that have no function Eukaryotic genes contains introns, normally intons are removed before mRNA moves to the cytoplasm. Bacteria do not have introns so may not have the appropriate enzymes to remove them, any protein translated will include the introns and won’t be functional Reverse TranscriptaseThough cells only have 2 copies of the insulin gene, there may be many copies of the mRNA strand, especially in those cells that secrete insulin. This mRNA can be extracted.The cDNA produced by reverse transcriptase is complementary to the RNA, therefore many copies of cDNA for insulin can be made.This doesn’t have the problem of introns as it is synthesised from the mRNA, where the introns have already been removed. DNA polymerase catalyses the synthesis of DNA that is complementary to the single stranded cDNA, making a double stranded molecule containing the gene for insulin.

Making Recombinant Plasmid

A bacterial cell won’t take in a gene spontaneously; it must be carried by a vector. In this case the vector is a plasmid, these move in and out the bacterial cells easily. Isolating Plasmid: To isolate the plasmid, the bacteria is treated with: EDTA-destabilises the cell walls Detergent-dissolves phospholipid cell membrane NaOH- creates alkaline environment to denature membrane proteins The plasmid is cut open using the same restriction enzyme as the one used to isolate the gene. The vector and gene are mixed and the complementary base sequences base pair with each other. DNA Ligase is used to bind the sugar phosphate backbones together. The gene has been spliced into the vector. The plasmid is now recombinant DNA.

Properties of a good vector: Self-replicating Small Not broken by host cell enzymes Doesn’t stimulate an immune response Can be screened Have markers to be identified in host

Transfer of DNA into Host Cell

To increase the rate of plasmids taken into the bacterial cells, calcium chloride is used, which binds to the negatively charged DNA backbone on the plasmid. The plasmid DNA passes into the cell with a heat shock, in which cells cooled to 4 °C are briefly heated to 42 °C.

Using Genetic MarkersSome bacterial cells do not take up the plasmid, some take up the unaltered plasmid, and some take up the recombinant plasmid. To differentiate between these, genetic markers are used.An unaltered plasmid has two genes for antibiotic resistance, they code to resist against two different antibiotics. An engineered plasmid has one gene for antibiotic resistance but the other gene has been cut, the gene which has been inserted into the plasmid has been inserted in the middle of this antibiotic resistance gene, rendering it useless.In this case the genetic marker is the antibiotic gene.For example if the unaltered plasmid has the genes to resist: peniciliin and tetracycline and the engineered plasmid has the gene to resist only penicillin. When a sample of the bacteria is grown on a growth medium containing penicillin both survive. However the bacteria with no plasmid dies. These colonies do not contain the plasmids.When a sample of these colonies is grown on growth medium containing tetracycline only the unaltered plasmid will survive as the engineered plasmids don't contain the gene to resist tetracycline., therefore the ones that die are the ones that have the engineered plasmid and so these colonies are taken and cultured.

The cells containing the recombinant plasmids are cultured in fermenters, each culture forms a clone. When the bacterial cells clone so do the plasmids.. The bacterial enzymes transcribe the insulin gene and translate the mRNA they produce. Insulin is therefore produced in large quantities and is purified for medical use.

Pros and Cons

Pros: Medical products: insulin, clotting factors to treat haemophilia, human growth hormone to treat types of dwarfism. These are safer then, which were purified in cadavers and could transmit disease. Prevention and treatment of disease: bacteria are modified to produce vaccines Crop Growth: modified molecules can secrete molecules that are toxic to plant pests

Cons: The antibiotic resistant marker genes can be transferred very easily to pathogenic species, which will then be untreatable with antibiotics Fragments of DNA, cDNA, mRNA may contain oncogenes that can be passed on to activate proto-oncogenes Micro-organisms with a new gene may become a threat if accidentally released A newly introduced gene may disrupt the normal function of other genes, in a way we do not currently understand