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pGLO Bacterial Transformation

Background and Student Protocol

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What did you notice?

About the bacteria under the UV light?

About the difference between the +pGLO and –pGLO bacteria?

www.bio-rad.com/glow

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E. coli colonies

The bacterial growth you see on the plate is made of millions of individual bacterial (E. coli) cells.

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Green Fluorescent Protein (GFP)

GFP allows us to visualize protein expression with UV light

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Genetic engineering using plasmids

plasmids

chromosome

bacterium

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Generic plasmid

gene

gene of interest cloned

into plasmid

pGeneric

ori

origin of replication allows bacteria to make copies of plasmid

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Bacterial transformation

Genetic transformation occurs when a cell takes up DNA and expresses the genes on that DNA.

Many different types of cells can be transformed – plant, animal, human, bacterial.

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Bacteria divide, and new bacteria get plasmids

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Genes are transcribed and translated

transcription

protein

translation

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Background Information

Antibiotic selection

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Plasmid with gene for GFP

gfp gene

encodes Green Fluorescent Protein (GFP)

ori

origin of replication allows bacteria to make copies of plasmid

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Plasmids can carry multiple genes

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β-lactamase gene for antibiotic resistance

amp^r (or bla) gene

encodes beta-lactamase,

an enzyme that breaks down

ampicillin (an antibiotic)

gfp gene

encodes Green Fluorescent Protein (GFP)

ori

origin of replication allows bacteria to make copies of plasmid

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Transformation with plasmid with amp^r

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β-lactamase produced

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Bacteria plated on ampicillin plates

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β-lactamase breaks down ampicillin

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Bacteria that aren’t transformed don’t grow

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Transformed bacteria grow into colonies

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Background Information

Control of gene expression with arabinose operon

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pGLO plasmid – regulation of GFP expression

amp^r (or bla) gene

encodes beta-lactamase,

an enzyme that breaks down

ampicillin (an antibiotic)

gfp gene

encodes Green Fluorescent Protein (GFP)

ori

origin of replication allows bacteria to make copies of plasmid

araC gene

encodes the

AraC repressor protein

pBAD promoter

landing site for RNA polymerase

to transcribe downstream genes

pGLO

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Without arabinose in the media

OFF

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With arabinose in the media

ON

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Arabinose operon (araBAD operon)

With arabinose, AraC undergoes a conformational change, allowing RNA pol to transcribe the araBAD genes

RNA polymerase

arabinose

ON

Three genes (araB, araA and araD) encode three enzymes involved in the breakdown of arabinose. They’re clustered together in what is known as the L-arabinose operon.

These three proteins are dependent on initiation of transcription from a single promoter, PBAD. Transcription of these three genes requires the simultaneous presence of the DNA template (promoter and operon), RNA polymerase, a DNA binding protein called araC and arabinose.

araC binds to the DNA at the binding site for the RNA polymerase (the beginning of the arabinose operon). When arabinose is present in the environment, bacteria take it up. Once inside, the arabinose interacts directly with araC which is bound to the DNA. The interaction causes araC to change its shape which in turn promotes the binding of RNA polymerase, and araB, A and D, are transcribed. Three enzymes are produced, they break down arabinose.

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Once arabinose is metabolized, switch is off

Without arabinose, AraC acts as a repressor – a loop is formed in the DNA, preventing RNA polymerase from transcribing araBAD genes

AraC

OFF

RNA polymerase

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Control of GFP expression - pGLO plasmid

Without arabinose, RNA polymerase is blocked from transcribing the GFP gene.

AraC

OFF

RNA polymerase

The pGLO plasmid has been designed to express green fluorescent protein (GFP), under the control of the arabinose operon (araBAD).

Scientists took inducible operons from bacterial chromosomal DNA and genetically engineered them into plasmids.

In the case of the pGLO plasmid, a team of scientists made a plasmid called pBAD18 by cloning the araBAD promoter and the gene for the AraC repressor protein into a parental plasmid containing an ori and the antibiotic-resistant bla gene.

Another team cloned the green fluorescent protein (GFP) gene from the jellyfish Aequorea victoria into a different plasmid.

A third group of scientists then used restriction enzymes to clone the GFP gene into the pBAD18 plasmid downstream of the araBAD promoter. As a result, the araBAD operon and the AraC protein regulate the expression of GFP. Using this system GFP is expressed only in the presence of arabinose.

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Control of GFP expression - pGLO plasmid

With arabinose, RNA polymerase can transcribe the GFP gene

ON

RNA polymerase

arabinose

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pGLO plasmid

amp^r (or bla) gene

encodes beta-lactamase,

an enzyme that breaks down

ampicillin (an antibiotic)

gfp gene

encodes Green Fluorescent Protein (GFP)

ori

origin of replication allows bacteria to make copies of plasmid

araC gene

encodes the AraC

repressor protein

pBAD promoter

landing site for RNA polymerase

to transcribe downstream genes

pGLO

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Background Information

Bacterial transformation steps

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CaCl₂transformation solution

plasmid

plasma

membrane

negative charges

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CaCl₂transformation solution

Ca²⁺shields

negative charges

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Incubate on ice for 10 min

Ca²⁺shields

negative charges

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Heat shock at 42°C for 50 sec

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Incubate on ice for 2 min

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LB broth

LB (lysogeny broth or Luria Bertani) broth is like chicken noodle soup for bacteria. It has all the nutrients bacteria need to grow:

Carbohydrates
Amino acids
Nucleotides
Salts
Vitamins

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LB recovery

During recovery, E. coli repair their cell walls and express the antibiotic resistance gene so they can grow on ampicillin plates.

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Student Lab Protocol

Bacterial transformation with pGLO plasmid

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Lab protocol

Label 1 empty tube – and the other +. Place tubes in foam rack.

1.

transformation solution

ice

TS

+

–

250 µl

Add 250 μl of transformation solution (TS) into each tube.

2.

Place both tubes on ice.

3.

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Lab protocol

Use a loop to pick up 2-4 large colonies of bacteria from the starter plate.

4.

Put the loop into the transformation solution in the – tube. Spin the loop between your fingers about 30 seconds, until the bacteria is completely distributed (no chunks). Close the tube and put it back on ice.

5.

ice

–

+

Using a new loop, repeat step 5 for the + tube.

6.

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Look at the plasmid under UV light

What color is the DNA?

Can you see any green?

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Lab protocol

Use a new loop to transfer a loopful (~10 μl) of pGLO plasmid to the + tube. Swirl the loop in the tube to mix. Close the tube and put back on ice.

Important! Do NOT put pGLO plasmid into – tube!

7.

Incubate both tubes on ice for 10 minutes.

8.

–

+

ice

10m

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Lab protocol

While the tubes are on ice, label agar plates:

LB plate: –
LB/amp plate: –
LB/amp plate: +
LB/amp/ara plate: +

9.

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Lab protocol

Heat shock. Bring your tubes on ice to the water bath. Set time for 50 seconds. Place the rack with the tubes in the 42°C water bath for exactly 50 seconds.

10.

10m

50s

2m

42°C

Put tubes on ice and incubate for 2 minutes.

11.

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Lab protocol

Move the tubes out of the ice and onto the benchtop.

12.

Close the tubes and incubate at room temperature for 10 minutes.

14.

10m

LB

–

+

Add 250 μl of LB broth (LB) to each tube. Use a new pipet for each tube.

13.

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Lab protocol

Flick the tubes a few times to mix up the bacteria.

15.

Add 100 μl (about 1 drop) of bacteria from each tube to the appropriate plates. Use a new pipet for each tube.

.

16.

–

+

100 µl

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Lab protocol

Use a loop to spread the bacteria all over the agar. Use a new loop for each plate.

17.

Stack plates upside down, tape together, and add lab group or initials. Incubate at 37°C overnight, or at room temperature for 2 days.

18.

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Results & Analysis

After Day 1

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Predict results

LB

–

LB/amp

–

LB/amp

+

LB/amp/ara

+

Ara

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Expected results

LB

–

LB/amp

–

LB/amp

+

LB/amp/ara

+

Off-white lawn of bacteria covering plate

No bacterial growth

Many off-white colonies (~75)

Many off-white colonies that glow green under UV light

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Model Recombinant GFP Bacteria

Day before transformation lab

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Use these images to design a model for how scientists made recombinant GFP bacteria. You can resize, copy and paste, recolor, or create your own elements. Include a written description of what is happening in your model by typing over these instructions.

gfp

gene

GFP protein

RNA

DNA

plasmid

E. coli

E. coli expressing GFP

Group 1

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Resources

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pGLO Resource Webpage

www.bio-rad.com/teachpglo

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Instructor manual and student quick guide