MCB2010C lab quiz #4

Understand the theory behind bacterial transformation

it is the uptake of naked (free) DNA molecules by competent (natural ability to take up DNA molecules) from their environment.

What is it meant to be competent?

To have the natural ability to take up DNA molecules from their environment. able to take foreign DNA

Procedure and reagents need it for bacterial transformation.

1. Place E. coli cells into 2 tubes of calcium chloride (CaCl2). Cells should be in log phase of growth

2. Add pGreen plasmid to one of the CaCl2 tubes (+pGreen tube). No pGreen is added to (-pgreen tube)

3. incubate tubes on ice (slows movement of cell membrane)

4. Heat shock: removed tubes from ice and immerse in 42 degree celsius water bath for 90 seconds (increases permeability of membrane)

5. return both tubes to the ice for at least 1 minute to allow bacteria to return to necessary temperature for survival.

6. Add LB broth to each tube (provides nutrition for growth and gene expression

7. plating: spread cells into plates

reagent is calcium chloride

What is the name of the plasmid. What genes were on the plasmid? Which is the reporter gene? Which is the selective marker gene?

The name of the plasmid is pGreen. It has 2 genes: ampicillin (phenotype: bacteria is able to resist and grow on bacteria on ampicillin selective marker gene) resistance and GFP (phenotype: green fluorescence aka reporter gene which shows if it has been transformed or not) .

What is considered special about the MM294 strain of E. coli used?

It is naturally competent (able to take in foreign DNA).

What role does CaCl2 play in bacterial transformation

Helps DNA to get in by masking/covering negative charges of DNA with positive charges. promotes dna uptake.

What is the purpose of heat shock and recovery

heat shock makes lipids move faster which allows more stuff in (foreign dna ) recovery is to keep it in and make sure it doesn’t die.

Why did we include ampicillin in the medium?

regular e.coli mm294 will not grow on ampicillin. E.coli mm294 has to be successfully transformed with pGreen plasmid then it will grow on amipicillin.

Identify positive and negative control plates, and experimental plates.

-pGreen LBA: not transformed- expect growth (positive control)

+pGreen LBA: transformed- expect growth (positive control)
-pGreen LBA+AMP: E.coli mm294 no plasmid - no growth (negative control)
+pGreen LBA+AMP: E.coli mm294 w/ plasmid (has been transformed with plasmid) - will grow (green fluorescent) (experimental plate)

• satellite colonies (green colonies on experimental plate)

Know the hypothesis, results, and explanations to all of these

lab 17

lab 17

What kind of radiation is UV? Nonionizing vs. Ionizing

UV is a nonionizing radiation.
- Excited electrons in molecules

How does UV light damage living cells? ( be specific)

UV light damages living cells by mutating DNA. Exposure to ultraviolet light causes adjacent thymine’s to become cross linked, forming a thymine dimer which disrupts normal base pairing.

What specific wavelength of radiation maximizes damage (UV-A, UV-B or UV- C)

UV-C Radiation. It is most effective at 264 nm.

What repair mechanisms do cells have?

The SOS system and Nucleotide Excision repair. Nucleotide excision repair is when nuclease comes and removes the damaged DNA (thymine dimers) and then DNA polymerase will come and add a new regular strand to that area and then DNA ligase will come to seal the fragments.

Which two bacteria were used in the lab?

Escherichia coli and Bacillus Megaterium.

Experimental design:

a. Why was a UV hood emitting light at 260 nm used?

b. What is the control in this lab?

c. What is the purpose of keeping the plastic petri lid on the plate during exposure?

a. because 260-264 nm is known to be the most effective rate to kill bacteria. (proper disinfectant)

b. The Control in this experiment was the half cover plates with index cards. (UV can not penetrate an index card)
c. In most cases, the lid should be removed from the petri dish to allow the UV light to kill the bacteria. The lid may make it difficult to kill bacteria

What did the results determine about sensitivity of these two bacteria to UV?

S. aureus can only requires have 5 min of UV radiation for it to be fully killed. B. megaterium requires 60 min for it to fully die.

How did their results differ? Why?

The results showed that S.aureus is an extremely easy bacteria to kill while Bacillus megaterium can take awhile due to its endospores.

What was the effect of leaving the lids on during exposure?

The clear lids blocked the UV light from killing the bacteria

lab 18

lab 18

Identify standard conditions of Kirby-Bauer Disk Diffusion Procedure

The Kirby Bauer disk diffusion procedure is the most widely used disk diffusion antibiotic susceptibility test. It determines what choice of antibiotics should be used when treating an infection. Standard conditions include:
- using Mueller Hinton II Agar (MHA)
- Media standardized with ph of 7.2-7.4
- Agar depth (4mm)
- needs to be 24 hour old bacteria cultures.

Identify steps of the Kirby-Bauer Disk Diffusion Procedure.

1. Use the sterile swab to aseptically transfer a proper amount of broth culture

2. Use the swab to create a lawn on the MHA (Mueller Hinton agar plate)

3. Allow the plate to dry for 5 minutes. Use a antibiotic disc dispenser (stored in the fridge) to dispense discs containing specific antibiotics onto the plate. Make sure to use flame sterilized forceps to gently press each disc to the agar.

What is a bacterial “lawn” and how is it created?

A bacterial lawn is when the entire surface of the agar is evenly distributed with the broth culture. It is created by aseptically transferring a swab of bacteria from a broth culture to a agar plate.

What is a “zone of inhibition”? What factors can affect the size of the zone of inhibition?

it is when antimicrobial agents diffuse into the surrounding agar and may prevent growth of the bacteria.
Factors:

• depth of the agar

• solubility and size of the antimicrobial compound.

Zones of Inhibition: How are the measurements taken and which metric units are the answers expressed in? How are zones of inhibition evaluated?

Zones of inhibition are determined by the R.I.S table (resistant, intermediate, and susceptible). Measured by using a ruler in cm but each cm = 10 mm. Measured in mm. They are evaluated based on the RIS system.

Know how to read a chart of antibiotics listing the zone diameters and correctly

look at lab manual

designate the bacteria to be: R, I, S to that antibiotic.

lab ppt and manual

Know what the designations of R,I, S stand for.

r - resistance I - intermediate S- susceptible

Be able to evaluate which antimicrobials would be effective for a given organism.

S (susceptible) would be the most effective. Wouldn’t use an R or I

Review the Clinical Connection for The Kirby - Bauer Method.

Wounded solider responded well to treatment but three days into his recovery his wound was infected and getting worse. Nurse swabbed the wound and sent a sample to the lab. They found out that the bacteria causing this was pseudomonas aeruginosa. Next antimicrobial samples are placed on a pseudomonas aeruginosa lawn and the zone of inhibition determines if the bacteria is good or not for this specific infection.

lab 19

lab 19

Know what PCR stands for.

Polymerase chain reaction (PCR)

Understand the theory behind and procedure for PCR. What does it accomplish?

“DNA photo copying machine.” used for sequencing, and diagnosis of hereditary and infectious diseases… The chain reaction is a 3 step process of denaturation, annealing and extension that is repeated in several cycles. After each cycle the number of copies is doubled. After completion of PCR, 1 billion copies of a DNA target sequence are produced.

Understand the events of PCR: Denaturing, Annealing and Extension

Denaturing: DNA gets melted at high temperatures

Annealing: join or stick

Extension: In the extension step, Taq polymerase synthesizes the new DNA strand by adding nucleotides (dNTPs) to the primer’s 3′ end. The DNA is synthesized in the 5′ to 3′ direction.

Know the purpose of Taq DNA Polymerase?

taq polymerase is heat resistant (from thermophilic bacterium) Purpose is extension. It is what copies that gene.

Know the purpose of Primers.

primers find the gene. locate the gene by binding to it (matching the letter to it on either ends)

Know how the locate the mecA gene on a finished Agarose Gel Electrophoresis .

if there is a line on 500 bp for each patient it means that there is the presence of a 533 bp band corresponding to the mecA gene (indicates MRSA)

What size band identifies the mecA Gene on a finished Agarose Gel Electrophoresis?

533 bp band

What are the advantages and disadvantages of using PCR instead of the Kirby Bauer Method?

PCR
advantages: PCR is much quicker
disadvantage: technology dependent, which limits how many people can do it.

Review questions at the end of Lab Exercise 19

lab manual