Module 6 Flashcards
genetic engineering
manipulating the DNA or genetic material of an organism to include the DNA from a different organism
plasmid DNA
small circular pieces of DNA that can replicate independently because they have an ori and can carry genes that encode for beneficial traits
easily transferrable
transformation
process by which bacteria acquire altered genetic characteristics from a different source
vector
serves as the vehicle for genetic material aka plasmid DNA
reporter gene
makes it possible to visually distinguish bacteria that carry a plasmid with foreign DNA from those that don’t aka GFP
competency
bacterial cell wall is made permeable to macromolecules such as DNA
can artificially be made competent by treating it with CaCl2 or abrupt transitioning between heat and cold environments
pGLO plasmid
contains genes coding for amp resistance and for GFP production
enzyme b-lactamase
secreted by transformed cells where it destroys ampicillin
arabinose operon
set of genes that code for enzymes that break down arabinose
those genes were replaced by GFP gene
presence of sugar= genes will be transcribed and GFP will be produced
ori
a sequence of DNA where replication is initiated
selectable marker
a gene that presents resistance to ampicillin, resulting in the production of b-lactamase
part 1
label one tube + and -
transfer 250 ul of CaCl2 into each tube and place both on ice
pre-incubation
pick up a single colony of bacteria from starter plate and immerse it into transformation solution in both + and - tubes and place them back on ice
incubation
get a loopful of pGLO DNA solution into + tube and mix it. none in - tube and then incubate tubes for 10 minutes on ice
Plates
LB/amp: +pGLO
LB/amp/ara: +pGLO
LB/amp: -pGLO
LB plate: -pGLO
Heat shock
put both tubes into water bath of 42˚C for 50 seconds then place them back in ice for 2 minutes
recovery and growth
remove tubes from ice and add 250 ul of LB nutrient broth to both tubes and incubate for about 10 minutes at room temp
Selection
pipet 100 ul of transformation onto each nutrient agar plates and use sterile loops and spread the suspension evenly
plates will be incubated for 24 hours
Transformation efficiency
number of transformed colonies/amount (in ug) of DNA used x final transformation suspension (ml)/volume (ml) plated
