LECTURE 7 - PLASMID VECTORS

what is a plasmid

a piece of self replicating, extrachromosomal DNA

what form are plasmids usually found in

supercoiled

what is the size range of plasmids

1000bp to 100,000bp

what is a crytpic plasmid

a plasmid whose use is unknown

list 6 plasmid types

general cloning plasmids

bacterial expression plasmids

mammalian expression plasmids

phagemid plasmids

cosmids

shuttle plasmids

what does recombinant selection refer to

the ability to determien which clones contain an inserted piece of DNA

what are our 3 main methods of recombinant selection

1. antibiotic resistance

2. lac Z

3. pZero type

how does antibiotic selection work

plasmids contain antibiotic resistance genes, so we insert our genes of interest into these plasmids

then we insert these plasmids into cells, so as a result the cells gain the antibiotic resistance

by using a plate containing the antibiotic, only the cells which successfully took up plasmids will survive

how does lac Z selection work

it involves the lac operon which codes for; B-galactosidase, permease, and transacetylase

B gal breaks down lactose, some of which becomes allolactose (allolactose allows expression by binding to the inhibitor)

on an x gal plate, B gal digestion will produce a bright blue colour

if we insert DNA then the alpha chain of B gal will not be made, so B gal is inactive

if no B gal is present then the X gal plate cannot be digested, and our colonies will be white.

why do we use IPTG in lac Z seelction

because IPTG will ensure the lac system operates by inducing it

what is pUC a derivative of

pBR322

what is derived from pUC

pGEM and pGEMT

what are pZERO plasmids derived from

pUC

hwo do pZERO plasmids work

the lac Z and ccdB are found next to eachother

ccdB controls e.coli replication by killing it

if a DNA insert goes in front of lac Z then both lac Z and ccdB will be disrupted

if ccdB is disrupted, then it cannot kill the e.coli and so the recombinant e.coli grow

what is a cos site

cohesive end

what do cosmids carry

large fragments

what feature do cosmids have

cos sites

what do cosmid cos sites allows

packaging of the cosmid into lambda phage particles in vitro

what is a phagemid described to be a combination of

combination of plasmid and filamentous phage particles

how does a phagemid behave

it behaves as a plasmid in e.coli, and in the presence of a helper phage it will be packaged into viral particles as ssDNA

what do bacterial expression plasmids allow

induced expression of cloned DNA

what strong feature do bacterial expression plasmids usually have

they have strong transcriptional terminators and stop codon

what is a his tag

a tag of 6 histidines which can be used as an optional purification element

what is T7 RNA polymerase gene under

it is under the lac promoter and inducer, requiring IPTG for expression to occur

what do mammalian expression plasmids contain

viral promoter upstream adjacent to MCS
viral origin of replication

polyA element downstream from MCS

what does transformation refer to

the addition of new genetic material to a cell, changing the phenotype

what are the 3 main methods by which we transform cells; with pros and cons

chemical (CaCl2 or RbCl2); low cost but low efficiency

electrical (electroporation); high efficiency but high cost

coated particles