Plasmids Flashcards
what is auxotrophy and why is it useful
auxotrophy is the inablility of an organism to sythesise molecules that it needs to live
it is useful to control the growth of transformed cells
what are the types of cloning vectors
plasmids
phages
cosmids and phagemids
viruses
transposons
yeast or bacterial artificial chromosome
what are the key factors for a cloning vector
modifiable
selectable
transferable
detectable
what are the structural features of plasmids
origin of replication
multiple cloning site
can be conjugative
high or low copy
maintainable and stable
what can determine plasmid compatability
origin of replication type
no two plasmids in one orginism can have the same type
what are some types of dna introduction
transformation - plasmids
transfection - eukaryotic cells
conjucation - some plasmids
transduction - phages and viruses
what is conjugation and the f factor
the f factor is the fuck factor, is fertility plasmid in ecoli
conjugation is the transfer of dna between cells using a pilus
what is competency and what methods can induce this
the ability of cells to uptake dna
cacl and heat shock
electroporation
how many genes does wt ecoli typically have and how much do cloning strains have
wt - 4500
cloning strains - 4200
what are the standard naming for genes
genes are names using three lowercase letters, one upper case letter, and numbers where known
gryE96
what are the [] used to indicate
if genes sit on a specific plasmid
what are the modifications in xl-1 blue
gryE96 - naldixic acid resistance
endA - endonuclease defficient
recA - recombinant defficient
delta lac - missing laczya operon
thiA - thiamin auxotroph
f[] - carried on the f factor
Tn10- transposon with tet resistance
proAB - proline biosythesis
laclq - overpromoted laczya repressor
delta(lacZ)M15 - lacz missing first 15 bases
whats the general process for investigating a gene of intrest
isolate gene - insert into plasmid - inserted into host
how can you ensure vector orientation
use two restriction enzymes
what are some purification markers for proteins
poly cis tags
biotin binding protein
maltose binding protein
glutathione S transferase
what is insertional inactivation
inserting a goi into a resistance gene so recombinant plasmids lose the resistance, and testing againt both types using replica plating
what is replica plating
using a velvet cloth to move colonies to new plates to test resistances
how are xgal and iptg used to distinguish transformed plasmids
iptg is used as a non digestable lactose analouge, to activate the laccl repressor, then lacz cleaves xgal to form an insoluble blue dye, the alpha subunit of lacz is disrupted in transformed plasmid so the colonies are blue not white
