genetic engineering Flashcards
what does dna ligase do
catalyses the joining of sugar and phosphate groups within the dna
what is electroporation
method for introducing a vector with a gene into a cell wall with electricity. the electricity makes the cell membrane more porous
what is a plasmid
small loops of dna in prokaryotes
what is recombinant dna
altered dna from genetic engineering. (rDNA)
dna molecules created in vitro by joining DNA with a vector molecule and transferring it into another organism
what is the difference between in vitro and in vivo
vitro- study outside of an organisms e.g. using a dish
vivo- test or experiment inside an organism
what are the 4 stages of genetic engineering
- identify and extract the gene
- transfer into vector(plasmid) using electroporation
- vector carries gene into a recipient cell
- recipient cell expresses the gene
how come genetic engineering is possible
genetic code is universal, almost all organisms use the same 4 bases and the same codons code for the same amino acids
what is synthetic biology
a new field of science created by genetic engineering
what does restriction endonuclease do
used to cut genes at specific base sequences.
what is reverse transcriptase
it is the reverse of transcription
binds to mrna and turns it into single stranded cdna- complimentary dna ready for dna polymerase
what is the different types of vector, and the mainly used one
plasmid- can transger dna to bacteria or yeast- main vector
viruses- transfer DNA to humans or bacteria
liposomes- fuse with cell membranes to transfer DNA into cells
how do scientists track the dna they want to use
markers
give some examples of markers and the function
marker function- allows tracking and identification of genes
fluorescent markers- fluorescent under uv light
how are desired genes isolated.
mrna is identified with the required gene
add reverse transcriptase to catalyse the forming of single stranded cdna (complementary DNA)
by addition of free nucleotides and DNA polymerase makes a double stranded complimentary DNA containing required gene
what happens after desired gene isolation
restriction enzymes cut the dna to create DNA sticky ends.
it is then multiplied by PCR and amplify the gene
how do you obtain a plasmid
bacteria mixed with restriction enzymes to cut the plasmid at specific sites to produce sticky ends, which are unpaired nucleotide bases
what is a sticky end
a cut end of a dna which has exposed unpaired nucleotide bases
what occurs once the plasmid has been cut and sticky ends are formed
free nucleotide bases, which are complementary to plasmid sticky ends, are added to the gene causing gene and plasmid to anneal.
catalysed by DNA ligase
what are the different ways to get the vector to enter the recipient cell.
heatshock treatment- make recipient walls more porous by altering temperatures and presence of calcium chloride. allows vector to enter through pourous membrane
electroporation- electrical pulse applied to cell to disrupt the membrane and allow recombinant vector to enter
(plants) recombinant plasmids can be inserted into a bacteria which infects plants and naturally inserts its genome into host cells genome.
where was the first endonuclease enzyme taken from
e.coli bacteria
what are the two ends which can be achieved through restriction enzymes
cut sticky ends and blunt ends.
sticky- exposed unpaired nucleotide bases which make annealing easy
blunt- non cohesive ends.
describe genetically modified bacteria in the production of insulin
1- produce gene from reverse transcriptase and DNA polymerase to create insulin gene in human dna
2- restriction enzymes can and isolate human insulin gene and cut plasmid to produce sticky ends. plasmid complementary nucleotides are added to the insulin gene so they can anneal
3- ligase enzyme catalyses the annealing of the gene and plasmid. producing recombinant plasmids.
4- e.coli bacteria is mixed with recombinant plasmids and heat shock and calcium cl ions cause e.coli membrane to become porous and to easily take plasmids.
5- ecoli then inoculated into a petri dish and incubated upside down to reproduce and grow insulin.
why are petri dishes incubated upside down
to prevent condensation forming and disrupting colonies of bacteria in the petri dish
what temperature would gm ecoli be incubated at
37 so it can survive in human bodies
what are the key enzymes used in genetic engineering
dna ligase
restriction enzymes
what are the 4 techniques of isolating desired genes for genetic engineering
reverse transcriptase and cdna
restriction enzymes such as endonuclease
how are dna copies produced by cutting dna
restriction endonucleases are used to cut at specific sites and produce sticky ends with unpaired bases.
dna ligase is then used to form phosphodiester bonds and join the sugar phosphate backbone when joining dna and plasmids
what gene isolation process involved producing fragments with sticky ends
isolating genes with restriction endonucleases.
how are genes inserted into a plasmid
desired gene cut with restriction enzyme. same restriction enzyme will cut the plasmid
both will have complimentary sticky ends.
gene and plasmid are joined by dna ligase to join the sugar phosphate backbone.
Plasmid is then inserted into a cell
how are antibiotic resistance cells used to test if bacteria has taken up the gene
replica plating.
plasmids contains two resistance genes.
the plasmid then becomes recombinant as insulin gene is inserted by restriction enzyme and ligase
to an ampicillin agar plate containing colonies of bacteria. two plasmids are added, recombinant and the normal plasmid
TO SEE WHAT PLASMID IS TAKEN UP
- get a new plate containing (tetracycline- substance which normal plasmid is resistant to, recombinant plasmid isnt).
- only 3 colonies grow on tetracycline agar. we want the ones that DONT grow on the tetracycline because it contains INSULIN.
The ones that dont grow obviously contain insulin
what is bacterial conjugation
microorganisms can naturally exchange genetic material in a process called conjugation.
