Lab 2

Quorum Sensing Lab
*Vibrio Fisheri (gram negative)
at low population density don't glow
at high population density express luciferase gene
luciferase breaks down ATP to produce energy to make light

Methods
Inoculated bunch of test tubes of sea water agar with V. fisheri
Then we let those incubate for just over 24 hours with lots of oxygen flow and at room temp (25C)
Observe test tubes to see if they glow? if they are then its high population density and express luciferase gene.
My group, then will streak MSA, EMB, and Nutrient Agar

Results
Did it glow or not?
Then results on agars:
MSA - grow?
Ferment manitol?
EMB - grow?
Ferment manitol?
Nutrient agar - grow?

Pseudomonas aeruginosa: quorum sensing phenotype = biofilm
biofilms: protein, carbohydrates, lipids (costly)

Specific product: rhamnolipids (lipid and sugar) snot balls
pyocyanin (protein) purple; smells like grapes

2 strains of P. aeruginosa:
wild type = normal (PA01)
JP2 mutant = doesn't have AI gene, but all other genes

Can we combine JP2 and PA01?
Hypo: If we provide AI to JP2. It will express the PA01 like phenotype (it will make biofilms)

PA01 = Broth - filled with AI

Protocol
centrifuge broth with PA01 growing in it and sterile filter to remove bacteria
prepare set of test tubes with varying concentration of AI - containing both: sterile and unused broth
inoculate each test tube with 0.1 mL of JP2 culture

Check Results on THURSDAY

Confirm that PA01, JP2 are both P. aeruginosa (gram stain OR streak on diff/selective media)

Find Est. # of bacteria in JP2 culture

Set of test tubes
0% AI - broth With JP2 bacteria (-) control
100% AI - broth (+) control - inoculated with PA01 plain PTSB broth

25% AI - broth
50% AI - broth
75% AI - broth

PTSB AI
100% 1mL 0% 0mL
75% .75mL 25% .25mL
50% .50mL 50% .50mL
25% .25mL 75% .75mL
0% 0mL 100% 1mL

lab
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