quiz 2: techniques in protein purification

What is the central dogma?

A theory stating that genetic information flows from DNA transcribes to RNA translates to Protein

What did watson and crick figure out?

the structure of DNA and the flow of the central dogma

What is GFP?

a fluorescent chemical compound that can re-emit light upon excitation

other fluorescent proteins can accomplish this as well

What is a chromophore? What does this have to do with GFP?

moiety that causes conformational change of the molecule when hit by the light

the way that the protein folds allows it to emit fluorescence

How can we use chromophores/GFP as a tool in molecular biology?

it is able to track where things/proteins bind

Where are the major, minor, and emission peaks of GFP?

Major: 395 nm

Minor: 475 nm

emission: 509 nm

What would using multiple fluorescent proteins in a sample tell us?

could help to track multiple biological taregets or structures (i.e. different structures in a cell)

Where does GFP come from?

A. victoria, a jellyfish

What are the steps of purification of GFP?

• Express GFP in bacterial cells

• purify via column chromatography

• analyze by gel electrophoresis

what does the ori do here?

origin replication, needed in bacteria to replicate plasmid

What does the ampr gene do?

Codes for beta-lactamase, which is an enzyme that breaks down antibiotics (like ampicillin)

What does the ampr promotor (or any constitutive promoter) do?

promoter that starts at the beginning of the gene, always turned on, is able to transcribe ampr gene at all times, selectable marker

what does the araC gene do?

Gene that encodes a regulatory protein that binds to the pBAD promoter, only when arabinose binds to AraC is the production of GFP switched on, creates a change that prevents RNA polymerase from binding

What does pBAD do?

binds AraC-arabinose and promotes rna polymerase binding and transcription of the GFP gene

What is transformation?

a procedure to force cells to take up foreign DNA, critical step in altering their genetic content

Do bacteria normally take up DNA?

only som do in a process called natural transformation

How can we get bacteria to transform?

In pGLO, we get bacteria to transform by using a calcium chloride solution and rapid heat shock

How do we know if we are successful in transforming the DNA?

If we see glowing green colonies on the plate

How do we force the bacteria to produce GFP?

we add arabinose sugar to their growth environment, i.e. we use LB/amp/ara plates

How do we know the bacteria are producing protein? Is it the same in every system?

If we are seeing glowing, the protein is being produced.

How do we force the bacteria to keep the plasmid in the cell?

By using the bla/ampr gene we can keep the plasmid inside the cell, which codes for the beta-lactamase protein

It maintains antibiotic selective pressure by adding an antibiotic to the growth medium

what is gene expression of eukaryotic cells and bacterial cellls good for? Which one is easier?

Producing a high quantity of protein

It is easier to purify the protein from bacteria

What steps do we have to take if we want to produce proteins in a bacterial system?

clone the gene into a vector (plasmid)

Transform plasmid into bacterial cells

Express the protein

Lyle and purify the protein

Aka transformation, isolation, and analysis

What is the structure of GFP?

a beta barrel with an alpha helix near the center, there are three amino acids in the center that interact to form the chromophore, which allows for the the protein to glow

Why is GFP an excellent tool in many areas of biology?

It’s ability to form an internal chromophore without requiring any accessory cofactors, gene products, or enzymes/substrates other than molecular oxygen

Describe what we did to transform the bacterial cells.

First, we prepared the cells to take up DNA by incubating the cells in CaCl2, which helps to mask charges on the DNA and the cells membrane

Next, we heat shocked the mixture, which helps open up the cell membranes to take the plasmid in

Next, we allow the cells to recover

Finally, we plate them and expose them to antibiotics, eventually we will see the growth of bacterial cells that took up the plasmids and the expression of other genes contained in the plasmid

What does beta-lactamase do?

A gene that confers antibiotic resistance to ampicillin. When expressed, this protein breaks down ampicillin so it doesn’t kill the bacterial cell. Therefore, any cell that has this plasmid

what controls the GFP gene?

A (Pbad) promoter, it will not allow expression unless the cells are grown in the presence of the sugar arabinose

Describe the arabinose operon

The genes araB, araA, and araD are contained in bacteria and each code for three enzymes involved in the breakdown of arabinose

What initiates transcription for the arabinose operon? What is involved in the transcription?

A single promoter, Pbad

DNA template (promoter and operon), RNA polymerase, araC, and arabinose

How is araC involved in the arabinose operon? Describe the process the arabinose operon goes through.

It binds to the DNA binding site where RNA polymerase goes, interacting with the arabinose that the bacteria takes up, which causes araC to change shape and helps RNA polymerase to bind to the DNA. Then the three genes in the operon (araB,A,D) are transcribed and translated to produce three enzymes that break down arabinose. When the arabinose runs out araC returns to the original state and transcription shuts off.

How does the pglo plasmid differ from the arabinose operon?

It contains both the promoter and the araC gene, but the three ara genes have been replaced by a single gene that codes for GFP.

Describe how arabinose effect the pglo plasmid producing GFP.

The GFP gene is only transcribed when arabinose is present, the araC protein binds to the arabinose, which allows for GFP to transcribe to mRNA, which is then translated to be be expressed and as the cells produce GFP, they produce fluorescent light.

What happens to the bacteria colonies when pGLO is present, but there is no arabinose.

The bacteria will live and grow, but they will not glow

How did CaCl2 help us transform the bacteria?

It helps mask the charges on DNA and the cell membrane

How did heat shock help us transform the bacteria?

It helped open up the cell membrane in order to take the plasmid in