in vivo cloning - use of vectors

in vivo cloning

by transferring fragments to a host cell using a vector

sticky ends

sequences of DNA cut by restriction endonucleases are called recognition sites

if this cut is staggered then a single strand with exposed nucleotides is left on the DNA strand (sticky end)

if the same REase is used then all fragments have complementary ends

once two ends pair up DNA ligase binds sugar-phosphate framework of two DNA sections to become one

why are sticky ends important

as long as the same restriction endonuclease is used, DNA of any organisms can be combined

preparing fragment for insertion

for transcription of any gene to occur, RNA polymerase must attach to the DNA near a gene on to a promoter region

nucleotide bases of promoter attach both RNA polymerase and transcription factors so starts transcription

in this way, to end transcription a terminator region binds and releases RNA polymerase at correct point

insertion of fragment into vector

once fragment has been cut and promoter/terminator regions added, it must be inserted into vector

REase in plasmid is same as for DNA fragment so sticky ends complementary

DNA ligase fuses DNA fragments and plasmids into recombinant DNA

introducing DNA into host cells (transformation)

plasmids with recombinant DNA must be reintroduced to bacterial cells

involves plasmids and bacterial cells mixing in a medium with Ca2+ ions which along with the temperature makes bacterial membrane permeable to plasmids

why do few cells take up plasmids

only ~1% of bacterial cells take up plasmids when mixed

some plasmids close without incorporating DNA fragment

some DNA fragments join ends to form their own plasmid

identifying plasmids

bacteria have evolved antibiotic resistance enzymes

gene for these found in plasmids, codes for ampicillin or tetracycline resistance

identifying plasmids using ampicillin resistance

ampicillin resistance gene is unaffected by the recombinant gene

bacterial cells grown in medium with ampicillin

those with plasmids will have resistance gene so will survive and those without plasmids won’t survive

ways to identify success of recombinant DNA

resistant to an antibiotic

make fluorescent protein which is easily identified

produce an enzyme whose action is identifiable

replica plating with tetracycline

the recombinant gene has cut the tetracycline resistance gene

when grown in a medium with tetracycline, this will identify the recombinant bacteria

however it will also likely kill the very cells containing the required gene

fluorescent markers

gene from jellyfish which produces green fluorescent protein is transferred into the plasmid being recombined

the gene to be cloned is inserted into centre of GFP gene so successful plasmids will not produce GFP or fluoresce

these will not be killed so can be used immediately

enzyme markers

gene producing lactase enzyme can be used

lactase turns colourless substrate blue so the required gene is inserted into this gene making it non functional

if the cells are grown on a culture with colourless substrate the successful colonies will not cause a colour change so can be identified