Lab Session 07/08/09

Stochastic Switch Team

Today, we identified the strains and prepared the spectinamycin solution.

We will use the E.coli strain numbered as 2386.

B. subtilis 168 strain is already in the fridge. To test our biopbricks ww will use gfp-rrnb integration vector. It uses spectinamycin as the antibiotic,

We solved 65mg of the spectinamycin in 13 ml of water, purified the solution and placed it into the fridge.

Prepared volume of the spectinamycin solution: 13 ml
Concentration: 5mg/ml

Sporulation Tuning/ Chassis Team

Today, we prepared Lysozyme stock solution in preparation for the recovery of our cwlD spores kindly sent to us by Anne Moir from Sheffield University.

Referring to Method A of the protocol, it states that there should be 200ug per ml of buffer solution present, and 10mM of L-alanine to initiate germination.

The following paragraphs shows the calculations of stock solutions made, and how much solution to add to obtain final concentrations desired.

Preparation

Lysozyme

We need:

200ug Lysozyme in 1000ul DI Water
0.2mg Lysozyme in 1ml DI Water

We made 0.5g of Lysozyme in 10ml of DI Water which we filter sterilised, where:

0.5g Lysozyme in 10ml DI Water
500mg in 10ml DI Water = 50mg Lysozyme in 1ml DI Water
50 000ug in 1000ul DI Water = 50ug Lysozyme in 1ul DI Water

Therefore, the amount of Lysozyme stock solution to add to a ml of buffer solution is:

50ug (Stock Solution) / 2ug (Desired Molarity) = 25
In order to obtain a solution with a final concentration of 2ug/ul, 
the stock solution needs to be diluted 25 times.
Desired volume = 1ml
1000ul / 25 = 40ul.

40ul of Lysozyme stock solution is needed per ml of buffer solution.

L-alanine

We need:

10mM L-alanine
0.01M L-alanine

We made 0.03L (30ml)of 0.05M L-alanine which we filter sterilised.

The amount of powdered L-alanine needed to make 30ml of 0.05M L-alanine solution is:

MW * Desired Volume (L) * Desired Molarity (M) 
89.09* 0.03L * 0.05M = 0.134g

Our final solution volume inclusive of L-alanine is desired to be 1ml. Therefore, the amount of L-alanine stock solution to add, such that the final concentration of L-alanine is 0.01M is:

0.05M (Stock solution) / 0.01M (Desired Molarity) = 5
In order to obtain a solution with a final concentration of 0.01M,
the stock solution needs to be diluted 5 times.
Desired volume = 1ml
1000ul / 5 = 200ul

200ul of L-alanine stock solution should be added to the buffer and lysozyme solution to make up approximately 1ml.

Final Volume = 1ml
Volume of buffer solution = 1000ul - 200ul = 800ul
Since 1ml of buffer solution requires 40ul of lysozyme stock solution,
800ul / 1000ul = 0.8
0.8 * 40ul = 32ul

Therefore, 800ul of buffer solution requires 32ul of lysozyme stock solution.

After Method A

After the addition of L-alanine to the solution, which would supposedly initiate germination, the solution was left in the incubator for 10 minutes
After 10 minutes, the eppendorf tube containing the solution was spinned down for approximately 1 minute.
- Note: Fill another eppendorf tube with water and put it on the opposite site to balance out.
The supernatant was removed from the eppendorf tube and the spores were resuspended in 1000ul of LB solution.
A serial dilution was performed.