BMB 212 PSU - Exam 2

What is a recombinant DNA molecule?

DNA that contains elements that allow the DNA to function and a gene of interest

What is ORI and why is it important?

origin of replication - allows independent replication outside of the bacterial genome

Why does the plasmid contain an antibiotic resistance gene?

(KanR, AmpR) Allows bacteria that contain the plasmid with the gene of interest to grow under conditions that other bacteria cannot - selects for bacteria that contain the plasmid

Why does the plasmid contain restrictive enzyme sites/multiple cloning sites (MCS)?

Allows for DNA to be inserted into plasmid by cutting specific DNA sequences (inserting the GFP gene)

Why does the plasmid contain elements that control gene expression?

So that we can control where and when the gene of interest is transcribed and translated (LacI)

Why does the plasmid contain a tag sequence?

Used to 'tag' the protein of interest, attaches extra molecules to the end of a protein (6 His tag - 6 histidines attached to GFP)

How was the pET30 plasmid cloned in this lab?

restriction ligation (DNA cut and paste)

Describe how restriction ligation was used to create the pET30-GFP plasmid:

1. pET30 plasmid is cut with restriction enzymes to produce sticky ends

2. Restriction endonucleases digest the cut DNA (Cleavage)

3. GFP gene is cut with the same restriction enzymes

4. DNA ligase binds cut GFP to the sticky ends of the plasmid (Annealing)

What is transformation?

bacteria take up DNA from the environment

Why are the bacterial cultures placed in a mixture of LB and Kanamycin?

To kill the bacteria that did not take up the plasmid and nourish the ones that did

List the steps of the mini-prep procedure that was used to purify the plasmid DNA:

1. resuspend cells from cell pellet in solution using a buffer

2. lyse cells with cell lysate to free all DNA (contains NaOH and SDS to denature nucleic acids and break cell membrane

3. neutralize lysate to prevent damage to plasmid DNA

4. centrifuge to separate cell debris and chromosomal DNA from plasmid DNA in supernatant (cell debris and chromosomal DNA collect in a pellet) centrifuge supernatant in spin filter - plasmid DNA will bind to the filter membrane

5. wash filter membrane with ethyl alcohol to remove impurities

6. centrifuge with elution buffer - releases plasmid DNA from the filter membrane

What was the restriction digest used for?

to determine which plasmids contain the GFP protein (restriction enzymes will cut plasmid, depending on the size of the fragments will show if the plasmid contains extra DNA - GFP gene)

What factors affect the distance traveled by DNA in an agarose gel?

DNA size and shape

- larger fragments travel less distance

- nicked circles travel less

- linear DNA travels slightly further than nicked circles

- supercoiled DNA travels farthest (pure plasmids often take this shape)

What was in the 4 tubes used in the restriction digest?

tube 1: Plasmid DNA (no enzymes)

tube 2: Xbal and Plasmid DNA

tube 3: EcoR1 and plasmid DNA

tube 4: Xbal, EcoR1, and plasmid DNA

What should each lane look like on the pET30 agarose gel? (restriction digest)

lane 1: DNA ladder

lane 2: one band ranging from 4-7 Kb (can be circular, linear, or supercoiled)

lane 3: one band at ~5 Kb

lane 4: one band at ~5 Kb

lane 5: two bands at ~4Kb and <0.5 Kb (may run off gel)

What should each lane look like on the pET30-GFP agarose gel? (restriction digest)

lane 1: DNA ladder

lane 2: one band ranging from 4.5-8 Kb (can be circular, linear, or supercoiled)

lane 3: one band at ~6 Kb

lane 4: one band at ~6 Kb

lane 5: three bands at ~4.5 Kb, ~1 Kb, and <0.5 Kb (may run off gel)

How does the Lac Inhibitor gene block the expression of GFP?

The Lac inhibitor gene (LacI) produces proteins in the absence of lactose that bind to the Lac promotor to inhibit the expression of the Lac operon (LacO) The LacO is next to the T7 promotor which controls the expression of the GFP gene

How are the cells induced to transcribe the GFP gene?

IPTG is a molecule that resembles lactose, which binds to LacI proteins and releases them from the Lac promoter and stops inhibition of LacO. LacO is transcribed and GFP is also transcribed because the promotor is at the end of the LacO gene

What are the two methods used to control gene expression of the GFP protein?

- T7 polymerase from bacterial genome

- LacI from plasmid

Why do we want to control gene expression of the GFP protein?

- too much energy use for gene expression can kill bacteria before gene of interest is transcribed

- protein may be toxic to cells (grow large numbers of cells to yield large quantities of protein)

What is leaky expression?

controlling gene expression does not turn a gene 100% on or off, cells that are not induced to express the gene can express it in small quantities

What is the relationship between the 6 His tag and Ni-NTA

The GFP protein is transcribed with a 6-histidine tag on the end of it. This tag binds to Ni-NTA beads so that the protein isn't lost in the purification process

List the steps of the GFP protein purification process:

1. Lyse cells with BugBuster to release protein

2. Protein his tag binds to Ni-NTA beads

3. Centrifuge to remove liquid (called flow through)

4. Wash column with wash buffer three times to remove other proteins

5. Elute protein with elution buffer twice which contains Imidazole - higher binding affinity for Ni-NTA - GFP is released

What is in the 8 protein purification tubes?

tube 1 (CL-UI): all proteins except GFP (may contain some GFP from leaky expression)

tube 2 (CL-I): all proteins plus GFP

tube 3 (FT): all proteins, no GFP (maybe some)

tube 4 (W1): leftover proteins

tube 5 (W2): leftover proteins

tube 6 (W3): leftover proteins - may be empty

tube 7 (E1): GFP (maybe some other proteins)

tube 8 (E2): GFP

What does the SDS PAGE look like for the purification process of GFP?

lane 1: protein ladder

lane 2: (UI-CL) lots of protein bands

lane 3: (I-CL) lots of protein bands; thick band of GFP at ~27 kDa

lane 4: (FT) lots of protein bands; no GFP

lane 5: (W1) lots of protein bands; no GFP

lane 6: (W2) less protein bands than W1; no GFP

lane 7: (W3) little to no protein bands; no GFP

lane 8: (E1) thick GFP band at ~27 kDa; may be other proteins

lane 9: (E2) thick GFP band at ~27 kDa