Genetic Engineering and Bacteria

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Genetic Engineering and Bacteria

Why do we want to genetically engineer organisms?

1.Improving a feature of the recipient organism; growth and resistance.               2.Engineering organisms that can synthesis useful products; insulin production, vitamin A production.               

Bacterial Cells and Plasmids are often used in genetic engineering -

  • Bacterial plasmids are used as a vector as the plasmid is a circular piece of DNA, They are separate from the main bacterial chromosome.
  • Plasmids often carry the resistance and antibiotic chemicals code.
  • When plasmids are cut with the same restriction enzyme, the complementary sticky ends will be formed.
  • Mixing the gene and plasmid with the ligase enzyme results in a recombinant plasmid.
  • Large quantities of the plasmids are mixed with bacterial cells which take up the recombinant plasmid,
  • Calcium Salts and heat shock is used ensures the plasmids are taken up by the bacterial cells.
  • Less than 1% actually take up the plasmid; transformed bacteria.
  • The bacteria contains new DNA; transgenic.
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Genetic Engineering and Bacteria

Bacterial conjunction and the advantages of taking up new DNA -

Bacterial calls are capable of conjunction; genetic material is exchanged. Copies of plasmid DNA pass between bacteria. The spread of the plasmids usually leads to spread of antibiotic resistance as plasmids often contain the resistant gene.

MRSA is a resistant strain of bacteria and can live on the skin. The advantage to the bacteria of conjugation is that it may give genetic variation and can survive in chemicals such as antibiotics.

Pneumonia in mice investigation - two strains of the bacterium;

  • S and R strain were used. S strain kills mice on infection. R strain does not kill mice on infection.
  • When injecting live R strain and dead S strain the mice still died, as R strain bacteria transform into S type bacteria.
  • R strain bacteria could take up DNA from S strain bacteria and produce the toxin found in the S strain.
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Engineering case studies -1: human insulin

Genetically engineered insulin -

  • mRNA of the gene from the correct pancreatic tissue was harvested and the enzyme reverse transcriptase was used to make the complementary DNA strand.
  • DNA polymerase and a supply of DNA nucleotides to the single strands of template DNA. Producing a copy of the original gene called a cDNA gene.
  • Unpaired nucleotides are added onto each end to give complementary sticky ends to the plasmid.
  • Plasmids are cut open by restriction enzymes and mixed with cDNA genes. DNA ligase enzyme reseals the gene into the plasmid .
  • The plasmids are now recombinant plasmids and mixed with bacteria that take up the plasmids.
  • The bacteria grow in culture and form a colony of identical cells.

Not all bacteria will take up the plasmid and still grow in this culture. Some may reseal to the plasmid they were before / some from bacteria that did not take up the plasmid.

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Engineering case studies -1: human insulin

Identification to transformed bacteria by replica plating -                     Radio labelled antibodies that bind to insulin was used in the past. Now plasmid vectors with genetic markers are used:

  • Original plasmids are resistant to two different antibiotic chemicals. These resistant genes are known as genetic markers. The bacteria are susceptable to both antibiotic chemicals.
  • The plasmids are cut by restriction enzymes at thier target sites in the middle of one gene that codes for resistance (tetracycline) so this resistance no longer exists, but the resistance for the other gene works (ampicillin).

Replica plating now used -

  • Bacterial cells are grown on agar so all bacteria colonies grow.Those transferred to the ampicillin agar that took up the plasmid will grow.
  • Some cells from these colonies are put onto tetracycline agar and bacteria that have taken up the insulin gene will not grow.
  • We can identify that any bacteria that grow on the ampicillin agar and not on the tetracycline agar have taken up the insulin gene.
  • These bacteria are harvested for large scale production of insulin.
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