Download Ch. 5A: Transforming Bacteria with Recombinant Plasmids

Ch. 5A: Transforming Bacteria with
Recombinant Plasmids
Learning goals
Describe the role of transformation in the gene
cloning process
Explain the purpose of each control in the
transformation experiment
Explain how the information encoded in a
gene is expressed as a trait
Key Ideas
A recombinant plasmid must be taken up by
bacteria
bacteria cell machinery used to replicate and to express
the gene of interest.
If transformed with the pARA-R plasmid bacteria
can be identified
Ampicillin will prevent the growth of cells that do not carry
an ampicillin resistance gene
Arabinose will activate the bacteria promoter that controls
expression of the rfp gene.
Go to pg 76 and complete
Lab 5A Transforming Bacteria with
the pARA-R Plasmid
RFP expression
Biotech Experience
araC gene
PBAD
Transcription
mRNA
Translation
araC protein
rfp gene
RFP expression
Biotech Experience
araC protein prevents RFP transcription by causing
a loop to form in the region of the r fp gene
araC gene
araC protein
PBAD
rfp gene
RFP expression
Biotech Experience
arabinose
Arabinose – araC protein complex prevents DNA looping
and helps to align RNA polymerase
on the promoter site (PBAD).
RFP
(red fluorescent
protein)
Translation
RNA polymerase
arabinose
araC protein
araC–protein
complex
mRNA
Transcription
araC gene
PBAD
rfp gene
Plating Tips
Note the plate markings: I=LB,
II=LB/amp, III=LB/amp/ara
Label the bottom of the plate near
the edge
Open the plates like clam shells
Sample goes on the agar, not the
lid
More Plating Tips
Agar is like jello, firm but not invincible, be
gentle – the “spreader is not a shovel
Turn the plates upside down (lids down) for
incubation, stacked and taped together
After incubation, do not open plates,
observe through the bottom
Remind Students
1. Sterile technique
Using bacteria
Contamination may affect results
2. Carefully READ and FOLLOW the lab
protocol.
Be sure lab partners communicate
3. No Food or Drinks
Sterile technique
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


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Always follow the protocol carefully – know
what you’re doing
Work quickly. Less time = Less opportunities for
contamination
Do not leave any container (tube, plate) open
any longer than needed
Watch what your equipment touches – there is
no “5 second rule” here.
All tips, tubes and spreaders go in the
“contaminated waste” container
• DO
NOT open plates, observe by viewing
through the bottoms
•Used plates – dispose in the “contaminated
waste” bags
P-
P+
LB
P-
P+
LB/amp
P+
LB/amp/ara
“oops” plates
Satellite colonies
Some cells without antibiotic resistance do
become "freeloaders" and survive because
other cells are doing the work of destroying
the antibiotic in their immediate vicinity on
the plate.
They only develop with antibiotics such as
ampicillin, that are destroyed by enzymes
such as beta lactamase outside of the cell.
Why do we get satellite colonies?

The ampicillin plate is old (meaning that the
antibiotic is partially degraded)
 The transformed cells are plated at very high
density (meaning that the plate is covered with
huge number of cells)
 The copy number of the plasmid in the cells is so
high that beta lactamase is secreted at high
levels,
 The colonies grow on the plate for several days
(allowing more time for degradation).