Question

2.What is the purpose (there’s two) of letting the transformed bacteria grow in recovery broth for a little while in 37 degrees before plating them on the selective media?

3.You are looking for a phage that will infect Burkholderia cepacia because it seems to be highly resistant to most antibiotics. You have the strain isolated in your lab so you can grow it up. You know it can cause onion rot, and is frequently found near tobacco or onion plants in the soil. Describe the steps you might take to isolate a novel phage from the environment that could infect Burkholderia cepacia.

Your answer doesn't need to be extremely detailed (you don't need to include specific volumes), just a couple sentences should suffice, but be sure to include any important details (i.e. if you need to make something sterile how do you do that)

4. For the bacterial transformation lab, what was the purpose of mixing calcium chloride solution with bacteria, and later performing a heat shock step after you've allowed the bacteria to sit on ice?

5. Giving animals too many antibiotics causes the animals to evolve resistance against the antibiotics.

True

False

6.

We went over two kinds of nucleic acid extraction. We went over "organic" RNA extraction where we use organic solvents to separate the RNA/DNA/protein/junk in a sample into layers or phases. In lab 3 you performed a DNA extraction but instead of the organic method you used a special column to collect the DNA. How did this work?

	a.	The silica column binds up the stuff we don't want, while the DNA passes through into the collection tube. We get any DNA stuck in the column out, by washing with ethanol into the collection tube.
	b.	The silica column binds up the stuff we don't want, while the DNA passes through into the collection tube. We get any DNA stuck in the column out, by washing with water into the collection tube.
	c.	We added a high salt concentration so the DNA would bind tightly to the silica column and everything else would simply wash through. We then eluted the DNA out of the column using ethanol.
	d.	We added a high salt concentration so the DNA would bind tightly to the silica column and everything else would wash through. We then eluted the DNA out of the column using water or 10mM tris buffer.

7. Beta-lactams target...

	a.	Protein synthesis (50S subunit)
	b.	RNA synthesis
	c.	Protein synthesis (30S subunit)
	d.	Cell wall synthesis

8. When we add Trizol (or phenol:chloroform:isoamyl alcohol, same concept) to extract RNA from a sample, the RNA is in the aqueous phase.

True

False

9.

Coronaviruses have plus-sense single-stranded RNA genomes (non-segmented). Rotaviruses have a genome divided into 11 shorter segments of double-stranded RNA. Say we had a sample that presumably contained both those viruses, and we wanted to make cDNA so we can sequence their entire genomes. Which of the following sounds like the best plan?

	a.	Destroy any contamination nucleic acid with DNAses/RNAses (viral RNA is safe in capsids so unharmed). Then extract the RNA from sample, mix a few microliters RNA with random primers, heat to 80 degrees for 5 minutes and place on ice, then add reverse transcriptase + RNAse inhibibitor+dNTPs+enzyme buffer, put back in thermocycler (25 degrees for ~10 mins), heat reaction to 50 degrees for an hour then heat to 70 degrees. Now you have cDNA, so you can prepare libraries for NGS.
	b.	Extract the RNA from sample, mix a few microliters RNA with random primers, reverse transcriptase, RNAse inhibibitor+dNTPs+enzyme buffer, heat reaction to 80 degrees for 5 minutes, place on ice, put back in thermocycler (25 degrees for ~10 mins), then let reaction go at 50 degrees for ~ an hour then heat to 70 degrees. Now you have cDNA, so you can prepare libraries for NGS.
	c.	Extract RNA, perform one-step RT-PCR by mixing RNA with reagents/enzymes for cDNA synthesis and PCR (RT and polymerase+ buffers+dNTPs, primers for both viruses). Then set up thermocyclyer machine starting at 45 degrees for the cDNA synthesis followed by regular PCR cycles. Sequence that product.
	d.	Extract the RNA from sample, mix a few microliters RNA with random primers, heat to 80 degrees for 5 minutes and place on ice, then add reverse transcriptase + RNAse inhibibitor+dNTPs+enzyme buffer, heat reaction to 50 degrees for an hour then heat to 70 degrees. Do a PCR (~40 cycles) on the cDNA you made using random primers and Sanger sequence that product.

10. We can infer that bacteriophages form a monophyletic group outside eukaryotic viruses (so all bacteriophages are more closely related to one another than they are to any eukaryotic viruses), because they all infect bacteria and bacteria are a monophyletic group.

True

False

11.

Which of the following tests would enable you to distinguish a microbe’s ability to ferment lactose?

	a.	Simmon's citrate Agar
	b.	Phenol red broth
	c.	Oxidase test
	d.	Gram stain

12.

You perform quantitative PCR using SYBR green dye (on the same DNA template/primers) in multiple wells. In one well ("well A") you get a Ct value of ~25. In another well ("well B") you get a Ct value of ~31.6. Which statement is most likely true based on this?

	a.	"Well B" had 6.1 times more starting template DNA than "Well A"
	b.	"Well B" had 20 times more starting template DNA than "Well A"
	c.	"Well A" had 6.1 times more starting template DNA than "Well B"
	d.	"Well A" had 20 times more starting template DNA than "Well B"
	e.	"Well B" had 100x more starting template DNA than "Well A"
	f.	"Well A" had 100x more starting template DNA than "Well B"

Answer 1

2.

When the transformed bacteria is grown in the recovery broth at 37 degrees, it allows the expression of antibiotic resistance genes from the acquired plasmid. Therefore, before plating the cells on antibiotic plates, this recovery step improves cell viability and hence cloning efficiency.

3.

Steps to isolate a novel phage from the environment that could infect Burkholderia cepacia:

a. Collect soil samples from different locations of the field where tobacco and onion plants are grown.

b. Culture the host bacteria (Burkholderia cepacia) in broth.

c. Add a given volume of a sample in the culture.

d. Incubate the culture for a day(18-24 hours).

e. Centrifuge and filter. This will remove most of the bacteria and the phage will remain suspended in the filterate.

f. Add chloroform. Remove the chloroform. (This will make the solution free of bacteria). You can use ultracentrifugation and then suspend in some buffer or autoclaved water.

g. Add a drop (10um) of this onto a bacterial lawn plate(Burkholderia cepacia). Incubate for a day.

Observe for plaque formation. Appearance of any plaque will confirm the presence of bacteriophage that infects Burkholderia cepacia.

4.

Calcium chloride is added for the divalent calcium cations. These divalent calcium ions creat transient pores in the cells walls and facilitate the entry of foreign DNA into the cells.

Increase in temperature increases the membrane fluidity, at high temperature, the bacterial cell membrane becomes more flexible and pores created by divalent cations becomes more efficient to serve as the entrance for foreign DNA.

5.

Giving animals too many antibiotics causes the animals to evolve resistance against the antibiotics. This statement is false. We give antibiotics to kill the infectious pathogen inside the animals and these antibiotics can become less effective if we give them too much of that because the microbe can evolve out of it.