A. survival of the plasmid inside the Lactococcus lactis, via the NICE vector
A. Changes in pH, temperature, and growth pressure will not effect the stability of the NICE vector inside Lactococcos lactis.
If we can't get the NICE vector into a MIXED population (mixed population on an antibiotic media (selective)
We need to show this vector containing strain is viable and competent with competition.
B. Spreading of the plasmid and antibiotic resistance
C. will the naked plasmid be uptake through transformation by other pathogens, following lysis of host cell.
Hints:
better to have over-controlled experimental outline than an under-control.
What is the best media to grow: M17 media, with a carb source: glucose (ours is lactose)
the vector is in the strain of Lactococcus. Change the
Where does Lactococcus stop growing (pH) and where is it most stable for replication? Test in the pH of a rumen stomach (5.7-7.3), temperature (37.8-40 degrees C).
Bacteria temperature test (bacteria may become cured of plasmid) grow in broth, to see if it'll last in the rumen of the animal.
Week 0
Make prep stuff: plates, broths, and frozen stalks, start growing plates and getting pure cultures to start (E. coli, B. subtilis, Salmonella, Typhe (typhoid), Camplyobacter, Yersinia enteraclidica )
Week 1
Plate the Lactococcus lactis and grow them over (x(get this from literature)) number of generations. This is going to extend beyond week one.
Broth culture and check heat stability. See at what point the plasmid gets cured, ie when does resistance get lost.
Growing some of the rumen strains with naked plasmid and testing rate of transformation ie see how much resistance is happening.