GCD Lecture 6

• PCR Inventor

  Kary Mullis
• antibiotic resistance genes

  AmpR: resistance to ampicillin (most common)

  CamR: resistance to chloramphenicol

  KanR: resistance to kanamycin
• Compare generated plasmid using gel electrophoresis

  Steps

  1. cleave the dna with restriction enzymes in five separate reactions
  2. separate the DNA fragments by 1% gel electrophoresis
  3. compare the dna fragment sizes on the gel to the sizes on the plasmid map
• When people tried expressing human proteins in bacteria, they found that the protein was rapidly degraded. When they made a fusion gene that produced a fusion protein that was part bacterial, the human protein wasn't degraded
• Part bacterial fusion protein

  use mRNA from fusion gene to make a plasmid (promotor, LacZ gene and somatostatin gene)

  mRNA gets translated into a fusion protein which is connected to amino acid, Met, and somatostatin polypeptide

  CNBr cleaves protein next to Met, resulting in fusion protein plus Met, and a separated somatostatin

  
• Insulin action and treatment

  action: a hormone that promotes glucose uptake

  treatment: for diabetic patients
• Factor VIII action and treatment

  action: blood clotting factor

  treatment: for certain types of hemophilia patients
• Biological control

  refers to the use of microbes or their products to alleviate disease or damage of plants from environmental conditions

  1. Nonpathogens: used to complete effectively against pathogens for nutrients or space
  2. microbes that produce helpful toxins: they inhibit other microbes or insects, but not the plant
• recombinant microbe

  one whose DNA is altered in lab
• Bioremediation/biotransformation

  use of microbes to reduce environmental pollutants (enzymes produced by a microbe transform the structure of the toxic pollutant)
• Making transgenic plants

  Gene of interest is inserted into the T dna of the Ti plasmid. The recombinant Ti plasmid is then transferred into A. tumefaciens. The T dna is transferred and incorporated into the plant cell chromosome. The plant cells are transferred to a medium to regenerate an entire plant (that does not carry gene for the wound).
• Alternative strategies to alter gene (transgene)

  DNA guns: shoot pellet coated with dna into cell

  Remove cell wall and get DNA in using conventional methods (electric current - punches holes in the cell and DNA will get in through there)
• Express human protein in animal milk

  Inject human hormone into sheep oocyte

  implant the fertilized oocyte into a female sheep, which then gives birth to transgenic sheep offspring

  obtain milk from female transgenic sheep

  purify the hormone from the milk
• Proteins that can be produced in the milk of domestic animals

  Protein and host

  • lactoferrin: cattle
  • tissue plasminogen activator: goat
  • antibodies: cattle
  • alpha, 1, antitrypsin: sheep
  • factor IX: sheep
  • insulin like growth factor: cattle
• Crispr-cas to make mutant mice

  microinject zygote into cell before it turns diploid (24 hour period), 75-85% of the mice will have the mutation
• In eukaryotes, when a protein being synthesized has an endoplasmic reticulum (ER) signal sequence, it is bound by an rna-protein complex called the signal recognition particle (SRP)
• SRP binding causes translation to pause
• the SRP binds a protein in the membrane that is next to a channel and this positions the ribosome. SRP lets go of everything and the newly made amino acid chain goes through the channel
• site-specific recombination

  two dna segments with little or no homology align themselves at specific sites (relatively short dna sequences that provide a specific location for recombo) the breakage and reunion of chromosome fragments is catalyzed by specialized enzymes
• site specific recombination is used by

  certain viruses when they replicate their genomes

  certain viruses and transposons to insert their dna into host cell dna

  the mammalian immune system to generate a diverse array of antibodies
• site specific recombo of bacteriphage p1

  cre recombinase catalyzes site-specific recombination between LoxP sites to produce circular single-copy genomes

  
• Use LoxP sequences to make a conditional gene

  cas9 protein cleaves at 2 sites

  HDR uses ultramer to ligate the DNA

  LoxP site on both ends

  A) LoxP site