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Lab 5a Transformation of Escherichia coli with pARA -R. “transformed cell” – cell has acquired new characteristics “characteristics” – due to the expression of incorporated foreign genetic material. The “acid test” – does it work?. The Big Picture. 2005 Pearson Education, Inc. - PowerPoint PPT Presentation
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Lab 5a Lab 5a Transformation of Transformation of Escherichia coli Escherichia coli with with pARA-RpARA-R
The “acid test” – does it work?The “acid test” – does it work?
“transformed cell” – cell has acquired new characteristics
“characteristics” – due to the expression of incorporated foreign genetic material
Differential gene expressionDifferential gene expression Gene expression – process by which the
information encoded in a gene is converted into an observable phenotype
Gene regulation – control mechanisms that turn genes on or off
Inducible proteins – synthesis is regulated depending on the bacterium’s nutritional status
Thank you Francois Jacob and Jacques Monod!◦Prokaryote operon model of gene control◦Repressors and activators are “trans-acting” – affect
expression of their genes no matter on which DNA molecule in the cell these are located.
Why do we need arabinose?Why do we need arabinose?
Why don’t we see the red protein in any LB growth media?◦Cells conserve energy and resources◦The rfp gene requires a specific substrate (arabinose) to be turned on (expressed)
RFP expression
rfp genePBAD
araC protein
araC gene
araC protein prevents RFP transcription by causinga loop to form in the region of the fp gener
RFP expression
rfp gene
PBAD
araC protein
araC gene
Rfp gene has replaced the araA, araB, and araD genes in the normal arabinose operon. These produce enzymes that digest arabinose.
araBaraA
araD
RFP expression
araC protein
arabinose
araC gene rfp genePBAD
arabinose – araC proteincomplex
RNA polymerase
Arabinose – araC protein complex prevents DNA loopingand helps to align RNA polymerase
on the promoter site (PBAD).
mRNA
Transcription
TranslationRFP
(red fluorescentprotein)
What do we know?What do we know?
1. Plasmid with gene of interest has been produced – confirmed by gel electrophoresis
What do we want to know?What do we want to know? 2. Can the plasmid (vector) be taken in by a host
cell (E. coli)? 3. Can the host cell express the gene of interest
and produce a product that can be utilized?
How is Lab 5a different from Lab 5?How is Lab 5a different from Lab 5?
“Materials” changes for “a” strand folks:
Instead of ‘E. Coli plate’ you will use:100 uL of competent cells
Instead of “1 mL 50 mM CaCl2” you will use:350 uL of LB broth (sterile)
Agar Plate tips to tell the students:Agar Plate tips to tell the students:
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(really, you need to remind them)
Agar is like finger 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
Three Important aspects to stress with Three Important aspects to stress with your students your 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 tips for studentsSterile technique tips for students 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 at the end of the lab.
Lab 5 day 2 • DO NOT open plates, observe by viewing through the bottoms•Used plates – dispose in the “contaminated waste” bags
LB
P- P+
LB/amp LB/amp/ara
P- P+ P+
Satellite coloniesSatellite 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?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).
Are satellites a problem?Are satellites a problem?
Probably not, provided that the colony of interest is subsequently picked and grown in fresh medium containing antibiotics.