Amgen Biotech Test

What is the difference between P+ and P- cultures?

The P+ tube contains recombinant plasmids and bacteria (and luria broth), and the P- tube only contains bacteria (and luria broth). The plasmid will give some of the bacteria in the P+ tube special traits: ampicillin resistance and the ability to produce rfp. The P+ cells will be able to survive when plated on ampicillin, and the P- cells will survive on luria broth plates. The purpose of the P- culture is to serve as a control. This step will confirm that our competent cells are alive and healthy, and not already resistant to ampicillin.

What is the purpose of growing transformed bacteria in the presence of ampicillin?

The ampicillin will kill of any E. coli that did not take up a plasmid. That way, we know all of the ones that form colonies on our plate contain the plasmid, and therefore will produce red fluorescent protein.

Binding Buffer

The binding buffer is used to unfold all the proteins so that the hydrophobic proteins stick to the resin and hydrophilic proteins pass through the column.

Elution Buffer

The elution buffer is poured into the column to release red fluorescent protein from the resin. All three buffers are needed because they all have a different concentration of salt which can be all used to separate proteins from each other.

If the gene of interest is controlled by an operon, such as the arabinose operon, when is the best time to turn on the gene?

The middle or the end of the log phase would be the best time to turn on the gene. At this point, there are lots of bacteria, because they've been putting all of their energy toward growing and dividing, but they are still all young and healthy.

What configurations (supercoiled, nicked circle, and multimer) might the plasmid have before digestion?

The plasmid could be all three: supercoiled, nicked circle, and multimer.

The Promoter Region (pBAD)

The promoter region is upstream of rfp, and it is essential for the rfp gene to be transcribed, because it is where RNA polymerase will bind.

Why do different plasmid configurations move the way they do through the gel? (Supercolied, nicked circle, and multimer)

The supercoiled plasmid is the most compact so it can go the furthest. The nicked circle takes up more space because it is not coiled, so it does not go as far. The multimer plasmid is two plasmids linked together, so it takes up twice as much space as the nicked circle. It will go slowly because it is very large and not compacted at all.

If pARA0R is digested with with BamHI and HindIII, what fragments are produced?

The two fragments that are produced are rfp with pBAD and Ara-C with ori and Amp-R. The rfp plus pBAD is 807 BP, and the Ara-c, ori, and Amp-R is 4495 BP.

Wash Buffer

The wash buffer is poured into the column to release moderately hydrophobic proteins from the resin.

Red Fluorescent Protein Gene

This gene codes for the the protein, red fluorescent protein, that we hope to purify in a lab.

The Ara-C Gene

This gene will make the E. coli which takes up our plasmid resistance to ampicillin. Then we can plate them on ampicillin to isolate only those that have taken up the plasmid.

The Ori Sequence

This sequence is where DNA polymerase binds, so if our plasmid has it, the plasmid will replicate inside its bacterial host and we will end up with more plasmids making the protein we want.

Once a gene has been inserted into a vector, what do you think is required to make the product encoded by the inserted gene?

Transcription and translation are required to make the product encoded by the inserted gene.

What will happen when bacteria cells that contain pARA-R plasmid are not given arabinose?

Without the arabinose, the RFP gene will not be expressed, so there will be no glow.

What is a plasmid?

A plasmid is a small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. Plasmids naturally exist in bacterial cells, and they also occur in some eukaryotes. Often, the genes carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance.

Why is a protein's conformation important for carrying out its function?

A protein is folded in a specific way to leave the correct binding sites exposed so it can interact with molecules.

What does P- contain?

Competent cells, luria broth

What does P+ contain?

Competent cells, recombinant plasmid, luria broth

Why does using two different restriction enzymes to cut the plasmid prevent the plasmid from reforming a circle without the inserted gene?

Cutting a plasmid with two different enzyme stops the plasmid from reforming a circle because the circle can't reform if the two different sides don't fit together. If it was only one enzyme, the two sites will just fit together eventually.

Why is it important to have multiple copies of a recombinant plasmid?

It is important to have multiple copies of a recombinant plasmid within a cell because the more plasmids there are in a cell means the more production and a higher chance of the genes being successfully passed on in a plasmid.

Why is it important to identify and verify a recombinant plasmid?

It is important to verify your plasmid to make sure that is is the size you expect it to be, that the restriction enzymes you use to cut it is where you expect them to be, and the correct size fragments result from restriction. If the plasmid or the fragments are the wrong size, it could indicate you have the wrong plasmid. If you're not starting with the right plasmid, you may not end up with the protein you're trying to get.

Why is it useful to use loading dye in this lab?

It is useful to use the loading dye in this lab because we can then see DNA move from one side to another on the gel.

What products might you expect to see in the R- and R+ tubes?

R-: supercoiled (5302 bp), nicked circle (5302 bp, multimer (10, 604 bp) R+: 807 bp fragment, 4495 bp fragment

Why did the red colonies only appear on the LB/amp/ara plate and not the Lb/amp plate?

Arabinose is nescessary to interact within the arabinose activator. The two together act as a transcription of rfp mRNA. Without arabinose, the activator is instead an inhibitor.