the expression of foreign genes in prokaryotes and eukaryotes

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  • Created by: Alex
  • Created on: 30-05-13 09:31
Why express foreign genes in cultured cells of whole organisms?
alter properties of crops or animal stock, alter properties of microorganisms, make useful proteins from bacteria, animal or plant cell cultures, to modify biochemical pathways & to make safe and effective vaccines
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What do you need to insert genes into another species?
1. Way of getting DNA into cells, 2. a way of maintaining DNA in the cell 3. A way of telling which cells in a population have incorporated the new gene, 4.a way of switching on and controlling th gene, 5. a way to ensure product is processed properl
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4 ways of getting DNA into cells (transformation)
1.Chemical treatment (Ca++) 2.Electrical discharge (electroporation-plant & bacteria) 3.pathogen (virus/bacterium -animals/plants/bacteria) 4.Injection into nucleus(animals&plants)
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what does electroporation do?
make membrane permeable
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2 ways of maintaining DNA in the cell
1. Attatch a replication origin to the gene inserted via a vector- so maintains self 2.DNA will be recombined into the chromosome-actively or passively
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to tell which cells in a population have incorporated the new gene you add a 2nd gene that
1. confers resistance to an antibiotic/lethal material 2.complement a mutation, e.g. can't make a.acids needed to survive, additional gene means it can make them
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Way of switching on and controlling the gene - promoter and control sequences
Must use sequences correct for the organism. In multicellular organisms, the enhancer must give expression in correct tissue type. In batch cultures, operator(bacteria) or upstream activating sequence(yeast) should be inducible (e.g.lac operon)
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A way to ensure that the protein product is correctly processed
Choose and appropriate host organism and tissue type.
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What is somatotrophin?
A peptide growth hormone
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Why can't somatotropin me extracted from human DNA using endonucleases?
Because gene contains 5 introns and has a human promoter and enhancer which won't work with e.coli
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How can we overcome problem or introns, promoter and enhancer?
lett the cell transcribe mRNA, and splice the introns out. Then make a cDNA from the mRNA
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What problems are there with the cDNA?
Still have a polyA tail and needs the addition of a prokaryotic promotor at one end, and terminator at the other
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where did somatotropin used to be sourced from and why isn't it sourced there now?
Brains of dead people. High chance of infections - prions
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why is position og 5' restriction site important?
distance between the promoter and start codon is critical for good expression
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What is the problem with the 5' enzyme sites?
enzyme A is too far away from translation start point, enzyme C would cut off part of the coding region, encoding for the first 20a.acids
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How was this problem got around?
DNA cut with enzyme C. this missing coding sequence was chemically synthesized including a enzyme C site and an artificial site for enzyme A was added to the 5'end. the cDNA and artificial fragment were joined by DNA ligase
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Expression cassette
Like an operon with several condisg cistrons removed artificial DNa consisting of recognition sites for several restriction endonucleases (a polylinker) inserted instead.
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Production of cDNA
1.enzyme reverse transcriptase starts 3'->5' elongation by adding deoxynucleotides to complement mRNA. 2.Product of reaction is hybrid molecule consisting of template RNA strand base paired with cDNA. 3.RNAase H nicks mRNA strand
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cont
4. at same time DNA polymerase1 uses fragments of nicked mRNA as primers to synthesis 2nd strand of cDNA, 5, finally and nicks in dscDNA are repaired by DNA ligase 6. result is double stranded cDNA
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What is in theE.coli vector?
A cassette, origin of replication and ampicillin resistance gene
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How did the cDNA get into the vector?
polylinker in the cassette cut with enzymes A and B and joined to the prepared cDNA
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How was the vector inserted into the bacterium?
Soaking bacteria in calcium chloride solution for one hour, mixing them with the DNa and heating briefly to 42 Degrees C
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How was the vector inserted into the bacterium?
Soaking bacteria in calcium chloride solution for one hour, mixing them with the DNa and heating briefly to 42 Degrees C
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what is the basic structure of a glycoprotein hormone family
2 polypeptide chains; the alpha subunit, which is the same in the 4 main proteins, and the beta subunit, which is different in each and responsible for the specificity of action
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why can luteinising hormone only be made in animal cells?
because either no glycosylation or the wrong type of glycosylation would occur
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problems with heat destroying foot and mouth for vaccines
too much heat it won't work as a vaccine, too little and the virus survives
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How does FMDV infect cattle?
Injects it's RNA into cells anr replies on the host ribosomes to translate it
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difference between a fusion cassette and a normal cassette
In a fusion cassette te restriction endonuclease is after the ATG inititiation codi
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Other cards in this set

Card 2

Front

What do you need to insert genes into another species?

Back

1. Way of getting DNA into cells, 2. a way of maintaining DNA in the cell 3. A way of telling which cells in a population have incorporated the new gene, 4.a way of switching on and controlling th gene, 5. a way to ensure product is processed properl

Card 3

Front

4 ways of getting DNA into cells (transformation)

Back

Preview of the front of card 3

Card 4

Front

what does electroporation do?

Back

Preview of the front of card 4

Card 5

Front

2 ways of maintaining DNA in the cell

Back

Preview of the front of card 5
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