molec lab 2 Flashcards
what will we be doing today
Bacterial Transformation using the pGLO system
BACTERIAL
TRANSFORMATION
he process by which competent
bacterial cells pick up DNA from the
environment and make use of the
genes it carries
An operon is a functioning unit of
DNA containing a cluster of genes
what controlls an operon
under the control of a single promoter
operons are mainly found in ____ but habve aklso been found in ___ and ___
prokaryotes: eukaryotes, viruses
Each operon minimally includes a
promoter
site
Operon regulation can be either _______ or ______ by induction or repression
negative or positive
Negative control involves the binding of a ______ to the operator to prevent
transcription
repressor
With positive control, an activator protein stimulates transcription by binding to _____ (usually at a site other than the operator)
DNA
Arabinose is a 5-carbon sugar in _____
E. coli cells
The arabinose operon consists of a promoter (PBAD) and three
structural genes which are
(araB, araA, and araD).
Without arabinose present,
RNA polymerase is unable to
bind to the promoter and begin transcription
of the genes.
The pGLO™ plasmid has been engineered to carry the arabinose
promoter and the gene for
green fluorescent protein (GFP)
PLASMIDS are
Small DNA molecule within a cell that can replicate independently
plasmids are most commonly found as
small circular, double-stranded DNA
molecules in bacteria
Artificially constructed plasmids may be used as vectors in
genetic
engineering
The pGLO™ plasmid has been
genetically engineered to carry a
replication origin (ori)
using a _____ transforming solution and heat shock to make
Escherichia coli cells competent
CaCl2
what does it mean when the switch is off?
When arabinose is not present, araC prevents RNA polymerase from binding to the promoter,
so transcription cannot occur
When arabinose is present, it binds to ___ and changes its shap
araC
what does it mean if the switch is on?
so RNA polymerase can bind to the promoter and transcribe the genes
After 10 minutes on ice, transfer the microtube holder/float (with both tubes in it) to the ___°C
water bath for exactly ___ seconds
42; 50 sec
This process of heat
shock makes the cell membranes more permeable to ___
DNA
No heat shock results in a ___ reduction in
transformants, whereas a 90-second heat shock yields about half the transformants
90%
