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strand 8 in vivo gene cloning (transferring fragments to a host cell using…
strand 8 in vivo gene cloning (transferring fragments to a host cell using a vector)
proteome - entire set of proteins that can be expressed by a genome at a certain time
use the same restriction endonuclease to cut the DNA fragment and the vector (carrier molecule) the sticky ends are complementary to one another and can combine DNA of one organism with that of any other organism
DNA ligase is used to bind the sugar phosphate backbone of the two pieces of DNA
roles of enzymes in genetic engineering
DNA ligase - seals gene into DNA of plasmid
restriction endonuclease - cuts fragments containing the desired gene from DNA at a specific sequence of bases called a recognition sequence
reverse transcriptase - turns RNA into DNA
RNA polymerase - joins nucleotides together
DNA polymerase - joins nucleotides together in DNA replication
promoter and terminator regions
before DNA is inserted into a plasmid (circular DNA found in bacteria that is separate from bacterial DNA) additional lengths of DNA are added
transcription - RNA polymerase attaches to promoter region along with a transcriptional factor of a gene
a promoter region needs to be added to DNA fragments if transcription occurs
a terminator region needs to be added to DNA fragment to stop transcription at the appropriate point
insertion of DNA fragment into a vector
a vector is used to transport DNA into the host cell
plasmids almost always contain genes for antibiotic resistance and restriction endonucleases are used at one of these antibiotic resistance genes to break the plasmid loop
the restriction endonuclease used is the same as the one that cut out the DNA fragment initially
this ensures sticky ends of the opened up plasmid are complementary to the sticky ends of the DNA fragment
DNA ligase joins the plasmids and DNA fragments - these plasmids now have recombinant DNA
identification
if plasmid was taken into bacteria it will also gtown on a medium containing an antibiotic as it will be resistant
bacteria without the plasmid won't survive
a restriction enzyme can cut a gene for antibiotic resistance and this can be used as a marker
a master plate is created
if plasmid is taken up with gene for antibiotic resistance would survive
marker genes: antibiotic resistance, fluorescent protein, enzyme with identifiable action
transformation
once DNA has been incorporated into plasmids it must be reintroduced into bacterial cells
this process is called transformation and involves mixing the plasmids and bacterial cells in a medium containing calcium ions
the calcium ions and changes in temperature make the bacterial membrane permeable, allowing plasmids to pass through the cell surface membrane into the cytoplasm