pGLO Plasmid and GFP Lab

What is a plasmid?

A small, circular, double-stranded DNA molecule distinct from chromosomal DNA.

What are the three main genes found on the pGLO plasmid and what do they do?

GFP (fluorescence), beta-lactamase (ampicillin resistance), and ara-C (regulates GFP transcription).

ARA-C regulator protein gene

regulates GFP transcription

GFP - Green Fluorescent protein

aequrea victoria jellyfish gene

Beta-lactamase

ampicillin resistance

What is the element responsible for active expression of the GFP gene? i.e. fluorescence

Arabinose sugar, which binds to araC to activate GFP transcription.

What are the experimental results of the media plates? i.e. what is observed in terms of bacterial growth

+pGLO LB/amp —> white colonies

+pGLO LB/amp/arabinose —> fluorescent green colonies under uv light

-pGLO LB/amp —> no growth

-pGLO LB —> bacterial lawn

Which plates should be directly compared to prove genetic transformation actually occurred?

The +pGLO LB/amp and -pGLO LB/amp plates grown on ampicillin.

What is the purpose of the calcium chloride transformation solution?

Neutralizes negative charges to facilitate plasmid entry into cells (make bacterial cells competent).

Why is the heat shock step precisely timed?

To temporarily create membrane pores for plasmid entry without killing cells (make bacterial cells competent).

heat shock

rapid change in temperature

Why must bacteria incubate in LB broth at room temperature for 10 minutes before being plated on ampicillin?

To allow transformed bacteria time to express the ampicillin resistance gene.

Luria Broth media

allows cells to recover

What is the name of the plasmid used in this experiment?

The pGLO plasmid.

What is the primary role of lysozyme in Part II of the GFP lab?

To enzymatically degrade the bacterial cell wall and lyse cells.

After lysing E.coli expressing the GFP, the sample is centrifuged, is the supernatant or pellet saved?

The supernatant containing dissolved GFP protein.

What type of chromatography is used to separate GFP from other proteins?

Hydrophobic interaction chromatography (HIC).

What type of electrophoresis is completed in the GFP lab?

polyacrylamide gel electrophoresis (PAGE).

polyacrylamide gel electrophoresis (PAGE)

confirm presence and purity of a molecule

What is the pore size of the gel controlled by?

Gel concentration (small proteins mirgrate more quickly)

Proteins

complex shapes and distinct charges that affect how proteins migrate through gel

How do scientists solve the protein problem?

Use chemicals that ensure all proteins are the same

What is the purpose of elution buffer in GFP chromatography?

To lower salt concentration and release GFP from the column by disrupting the hydrophobic interaction.

When is binding buffer used in GFP chromatography?

To maintain high salt and expose hydrophobic regions of proteins for the GFP to bind to.

What is the purpose of Laemmli buffer?

To denature proteins and impart a uniform negative charge.

Laemelli buffer

62.5 mM Tris buffer

10% glycerol

2% SDS (sodium dodecyl sulfide)

5% DTT (dithiothreitol)

0.01% bromoplenol blue

SDS

detergent used to disrupt interactions between AA

linerize protein

primary structure

standardize charge density

negative charge to proteins, specifically each AA

DTT

reducing agent, disrupt disulfide bonds

How does the plasmid in the pGLO experiment enter bacterial cells? Hint: 2 elements important

Calcium chloride treatment followed by a precisely timed heat shock.