Bio practical 2
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The pGlo plasmid contains:
- bla (codes for Beta Lactamase)
- araC (codes for araC protien)
- GFP (codes for Green Fluorscent protein)
Which plates show green color under UV light if pGLO plasmid is successfully transformed to baterial? (TWO answers 1')
C. LB+Ara D. LB+Amp+Ara
3. What chemical is used for transformation? (1')
4. Ampicillin interferes with bacterial growth by inhibiting?
Glucose and arabinose are added to media plate, which one will be first used up by bacterial regarding to existence of catabolite repression?
What is arabinose operon?
- AraC protein binds with arabinose, resulting in DNA confirmation changing that could allow RNA polymerase to bind GFP or BAD promoter and initiate GFP or BAD expression.
- Arabinose presence promotes GFP or BAD expression
- Arabinose absence inhibits GFP or BAD expression
Explain the purpose of heat-shock in transformation. (2')
Increase cell membrane permeability and allow DNA or plasmid to enter cell
Simply explain why the E. coli with pGlo+ plasmid will glow on plate LB+amp+ara, but will not glow on the plate LB+amp+ara+glu.
- Arabinose promotes GFP expression in LB+amp+ara
- In LB+amp+ara+glu, bacterial preferentially use glucose, which repress AraC expression and GFP expression.
For the transformation exercise, we used three different kinds of plates: LB; LB+amp; LB+amp+ara. Explain the function of each plate. (3')
- LB: control
- LB+amp: see if pGLO is successfully transformed to cell. If yes, the bacterial can grow on the plate, if not, bacterial can not grow
- LB+amp+ara: see if GFP expression can be induced by arabinose. If yes, green color under UV. Otherwise, no green color.
What functions for CaCl2 in heatshock?
- 1. CaCl2 treatment on ice crystalizes fluid membranes and stabilizes distribution of charged molecules
- 2. CaCl2 Transformation solution provides Ca++ cations that neutralize the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane, allowing the DNA to enter the cells
Plasmid-containing bacteria with desired traits to be passed on to other bacteria are considered? (1')
For a genotype to change, the DNA sequence must be altered. True or false?
After conjugation has occurred, donor cells will lose the plasmids and recipient cells will have the plasmids. True or false? (1')
Facts about conjugation
- involves pilus
- plasmids are typically the DNA transferred
- need direct cell-to-cell contact
- a plasmid is transferred differently than transformation
5. A strain that is auxotrophic for losing the ability to make an amino acid could NOT grow on? (1')
- minimal medium plate
- could grow on TSA, enriched medium, or complete medium
Differentiate Fitness and adaptation. (2')
- Fitness- the ability of an individual to produce offspring.
- Adaptation- a trait that increases the fitness of an individual in a particular environment.
Differentiate phototroph and auxotroph. How can they grow on enriched medium plate and minimal medium plate? (4')
- Phototroph: wild-type bacterial; can grow on both complete medium plate and minimal medium plate
- Auxotroph: mutated bacterial; can only grow on complete medium plate and cannot grow on minimal medium plate
What the three possible scenarios would happen to bacterial DNA after UV exposure? (3')
- 1. No damage to bacterial cell’s DNA
- 2. Induced formation of T-T dimers; damage repaired by DNA repair pathways
- 3. Induced formation of T-T dimers; not repaired; possible mutation results
What outcomes would be caused regarding overuse of antibiotics? (3')
1. A bacterium that carries an antibiotic resistance gene on a mobilizable plasmid, in the presence of antibiotic transfer of the mobilizable plasmid will be favored
2. Normal flora with mobilizable plasmid that encodes for an antibiotic resistance gene, might conjugate with pathogen bacteria and enable pathogen bacteria to acquire antibiotic resistance.
How could we increase the number of mutations regarding exposure to UV? (3')
- 1. Increase number of colonies
- 2. Extend time of exposure to UV or other mutagens.
Descriptions about coliforms
- aerobic or facultative anaerobic
- ferment lactose
- not pathogens, but can be opportunistic
- bacilli gram negative
Following characteristics of the skin make it an inhospitable environment for bacterial growth, except (1')
3. What enzyme is indicated in a positive MUG plate? (1')
4. Most of Gram negative bacteria can grow very well on Salmonella-Shigella agar plate. True or false? (1')
Gram positive bacteria can grow very well on Eosin Methylene Blue agar plate. True or false? (1')
Sort the ability of hemolysis for α, β, and γ types from strong to weak
β > α > γ
What is the full name for MRSA? How to treat MRSA? (3’)
- MRSA : Methicillin-resistant Staphylococcus aureus
- Treatment: vancomycin or wound debridement
The positive MPN (multiple tube technique) is
Yellow color + gas
What are the three stages in multiple tube technique for detecting coliforms?
- 1. Presumptive test
- 2. Confirmed test
- 3. Determination of the # of coliforms
According to EPA recommendations for certain types of water, the MPN index for body-contact recreation water should be fewer than 100 per 100ml of water sample. A student used multiple tube technique to test if the water of a lake can be used for body-contact recreation. His test results are: 3 positive for 10ml tubes, 2 positive for 1.0 ml tubes, and 1 positive for 0.1 ml tubes.
Questions: 1. Can the water of this lake be used for body-contact recreation? Why? (2’); 2. Which tube will be chosen for confirmed test? Why? How to identify it might be contaminated by feces?
- 1. The MPN index is 150 per 100ml. 150>100,
- So, it can not be used for body-contact recreation.
- 2. 0.1ml tubes will be chosen for confirmed test, coz it has the highest dilution.
- Inoculate the bacterial in this tube to MUG plate and incubate the plate under 37℃ for 24 hours. If florescent under UV light, it is a positive confirmed test， indicating a possible fecal contamination.
Large intestine has more bacteria than those in Stomach and small intestine?
Many of non-lactose fermenters in large intestine are non-pathogens?
Large intestine consists primarily of _____?
How many bacteria /ml indicates a UTI?
Which is not associated with detecting infection of lower-small and large intestine
- (you can use stool, rectal swab, or blood cultures)
differentiate mutualistic and antagonistic
- Mutualistic: mutually benefit
- Antagonistic: one represses the others
What diseases could be caused by urinary infection? Please also show the infected place in urinary tracts. (4')
- Cystitis : Inflammation of the urinary bladder
- Urethritis: Inflammation of the urethra
- pyleonephritis: Inflammation of the kidneys
List some causative agents and symptoms for the urinary tract infection.
- Agent: Mostly gram negative bacilli, esp E.coli, can also be caused by gram positive
- Symptoms: Pain, burning, frequency, urgency.
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