What is a restriction enzyme
restriction endonuclues is bacterial enzyme that recognizes a speicifc DNA sequence and cuts the DNA backbone
How does restriction enzme know to cut host DNA
using restriction modification, DNA methyltransferease methylates the host recognition sites, restriction enzymes cant cut methylated DNA, invading phage DNA is unmethylated and is cut
What are sticky ends
short single stranded overhangs produced when restriction enzymes cut Dna.
Why sticky ends matter
Complementary sticky ends can base pair, increase efficiancy of DNA ligation
what are 3 ways to reduce self ligation when making recombinant vector
Remobe the 5’ phosphate from the vector, directional cloning using diff restriction enzymes, blunt end cloning when compatible sticky ends are not available
why is lacZ placed upstream of restriction sites in insulin production
lacZ allows high level expression via lac operon, produces fusion protein that is stable , protects insulin from degradtion
What are the 5 main regions of Ti plasmids
T-DNA - transfered into plant genome can nly enter plant cell, Virulence genes = mediate dna transfer, opine synthesis genes, opine catabolism genes, origin of replication
relationship between agrobacterium tumefaciens and plants
tumefaciens cause crown gall disease, transfers tdna into plant genome, plant produces opines, bacteria uses opines as carbbon source
how does Ti plasmid re engineer for transgenic plants
using binary vector system, disarmed helper plasmid= virulence genes only, TDNA plasmid= gene of interest + selectable marker
why are most plants totipotent
single plant cell can regenerate entire organism, allows whole plants to be regenerated from transformed cells , clone themselves easily
how was dolly the sheep cloned
nucleus from adult mammary cell imserted into enucleated egg. embreyo implanted into surrogate mother
why is dolly not a true clone
her mitochondrial dna came from thr egg donor , only nuclear dna was cloned
concerns about transgenic plants
allergies to recombinant [roteins, spread of insecicide resistance in natural insect populations, tra sfer of herbicide reisstance to wild plants, negative impacts on natural inset pollinators
