Genetic engineering

Genetic engineering

  • the alteration of the genetic code by artificial means

Examples

  • banana vaccines

    • Produce virus proteins

    • Not the infectious parts

    • Eating banana triggers immune response

  • Venomous cabbage

    • Altered to produce scorpion venom that is harmless to humans but kills caterpillars that try to eat it

  • Insulin

    • Insulin for diabetes was first extracted from animal pancreas

    • Some people had allergic reactions to animal proteins

    • Now made using genetically engineered bacteria

Production

  • isolate the gene to be transferred

  • Add the gene to new cells

  • Identify the organisms that have had the new genes successfully added

Isolating a gene

  • cut the gene out of a length of DNA using restriction enzymes OR

  • Make DNA from RNA present cells

Restriction enzymes

  • REs cut DNA at a particular series of bases

    • Recognition/restriction site

  • Most recognition sites are 4-8 bases long

  • They are palindromic

    • GTATAC

    • CATATG

  • lengths of DNA cut with restriction enzymes are called restriction fragments

  • Different pieces of DNA cut with the same restriction enzyme will have complementary sticky ends

  • Could then be joined together

DNA from RNA

  • making a gene from RNA

    • A gene that is active in a cell will make a protein product

    • It must be copied into mRNA first before the protein can be made

    • This mRNA can be isolated from cells and copied back into a DNA gene

  • reverse transcriptase copies single stranded mRNA into cDNA (complementary DNA)

  • This can be made into double stranded DNA

Adding the new DNA to a vector

  • a vector is used to transfer the isolated gene that makes the product we want into the host cell

    • Insulin gene into bacteria

  • Types of vector

    • Viruses

    • Plasmids

  • Cut out gene of interest using RE

  • Cut plasmid with same RE

  • Sitcky ends anneal by hydrogen bonding

    • Gene is inserted into plasmid

  • Repair sugar phosphate backbone using DNA ligase enzyme

    • Forms covalent bonds between nucleotides

Marker genes

  • these are genes already present in the plasmid that allow the plasmids that have taken up the gene correctly to be identified

Transformation

  • once the gene has been added to the plasmid the plasmid must be inserted into a bacterium

    • Heat shock after incubation in cold CaCl2

    • Electroporation

  • However DNA is not always inserted correctly into the vector

  • Not all bacteria transform

  • Need to be able to identify those that have had the modified vector correctly inserted

Heat shock

  • calcium and an increase in temperature cause the membrane to become permeable so the vector can enter the cell

Electroporation

  • small electric current is applied to the bacteria

  • Membranes become very porus so plasmids can enter the bacterial cells

Genetic markers

  • used to identify the bacteria with correctly added gene and vector

  • Checks:

    • Has the gene been added to the vector

    • Has the vector been added to the bacteria

  • if no vector is inserted then white colonies will also form

    • Use antibiotic resistance to identify the bacteria containing the vector

    • Bacteria with no vector will not grow on agar with antibiotic as it does not have the inserted resistant gene

  • This can be carried out at the same time as lacZ