pglo experiment what is ampicillin? a cell wall inhibiting antibiotic what happens to normal...

Pglo experiment

• What is ampicillin?• A cell wall inhibiting antibiotic• What happens to normal bacteria that are grown on

agar plates with ampicillin in the agar?• The bacterial cells won’t grow on the agar.

Pglo Experiment (continued)

• What is beta-lactamase?• An enzyme that breaks down ampicillin.• What happens to bacterial cells that produce the

beta-lactamase enzyme (because the have the beta-lactamase gene) when you grow them on ampicillin agar?

• They grow!

Pglo Experiment (continued)

• What is GFP?• A green fluorescent protein (it glows green under UV light)• Where is the GFP gene located in our experiment?• On the pGlo plasmid

Pglo Experiment (continued)

• How do we manipulate Ecoli to take up the pglo plasmid (become transformed)?

• Heat shock• Osmotic shock (transformation solution)

Pglo Experiment (continued)

• What is ara?• A sugar that must be present in order for the

GFP gene to be expressed and form a GFP protein.

Pglo Experiment (continued)We have 4 plates in this experimenta. - DNA LBb. -DNA LB/ampc. + DNA LB/ampd. + DNA LB/amp/araWhat is the purpose of the –DNA LB plate?Which plates should be compared to determine if the bacteria have acquired the amp resistance gene?Which plates should be compared to demonstrate that arabinose is required for the expression of the GFP gene?Which plate is the control for the + DNA LB/amp/ara plate?

Pglo Experiment (continued)

• What do these results tell you about bacterial transformation with the Pglo plasmid

Employee Resource Manual

• Plasmids• Restriction Endonucleases• Agarose Gel Electrophoresis

Plasmids

• Small extrachromosomal pieces of DNA found in some bacterial species

• May carry additional genes (such as antibiotic resistance)

• Can be genetically modified and used as vectors for genetic engineering

PUC 18-Plasmid

What is Lac Z?

• It is the gene that codes for the enzyme B-galactosidase

• What does B-galactosidase do?• It hydrolyzes sugars including lactose and X-gal

What is X-gal?

• An artificial sugar that some bacteria can hydrolyze• What determines whether a bacterium can hydrolyze

X-gal?• The bacterium must have the enzyme B-

galactosidase• How can we tell if a bacterium has hydrolyzed X-gal?• The bacterial colonies will turn blue. If they

don’t hydrolyze X-gal, they will remain white.

Restriction Endonucleases

• Produced by some bacteria as a defense against virus infection

• Cleave DNA at specific bases sequences (different recognition site for each different enzyme)

• Can be used to join DNA from 2 different sources (plasmid DNA and genomic DNA)

ECOR1

Agarose Gel Electrophoresis

• Separates DNA based upon size differences• DNA is pulled through a gel by an electric

current• (-) charged DNA is pulled to the positive pole

of the apparatus.• Smaller pieces of DNA migrate through the gel

faster than larger pieces of DNA

Agarose Gel Electrophoresis

Procell

Procell in Action

What is your first job assignment?

• Clone the H gene (use a bacteria to make copies of the gene for us)

What kind of bacteria do we use to clone the H gene?

• E.coli (lacZ(-), amp sensitve)

Where is the H gene located?

• Lambda virus

How do you get the cloned gene into the bacteria so the bacteria can copy it?

• Transform E.coli (lacZ(-), amp sensitve) with PUC 18-lambda plasmid (heat shock and osmotic shock)

Lambda virus genes havebeen inserted into the plasmids here

How do you get lambda genes into PUC 18 plasmid?

• Incubate PUC-18 and lambda with EcoRI, ligate products

How many different plasmids do you get when you mix PUC 18 and lambda, both of which have been ECOR1

and then ligated?

7

Would all 7 of the plasmids be recombinant (have lambda DNA)? No!

How do you tell if bacteria have been transformed successfully with PUC-18 plasmid?

• They will grow on amp agar.

How can you distinguish whether plasmids that transformed bacteria were recombinant (lambda and

PUC-18) or nonrecombinant (pUC-18 only)?

Plate the transformed cells on Xgal-amp agar

E.Coli transformed With nonrecombinant Plasmids (PUC-18)

E.Coli transformed With recombinant Plasmids (PUC-18/lambda)