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

The Big Picture

2005 Pearson Education, Inc.

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)

pARA-R construct

Recombinant plasmid of interest

pARA-R

BamH I

Hind III

rfp702bp

4720 bp

RFP expression

araC gene rfp genePBAD

Transcription

mRNA

Translation

araC protein

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+

“oops” plates

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.