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Formal Lab Session - 12th August 2009

The Stochastic Switch team and the Chassis Team discuss ideas with Neil

Overview

Metal Sensor Team

Recap

In the last session the Metal Sensing team attempted to transform B. subtilis with pGFP-rrnB plasmid (an integration vector). We carried out the procedure and plated the 'transformants' onto 4 plates:

• LB alone - (MM competence medium MINUS B. subtilis)
• LB + Chloramphenicol - (Bacillus subtilis + water (NO DNA))
• LB + Chloramphenicol - 50ul of B. subtilis + pGFP-rrnB
• LB + Chloramphenicol - 200ul of B. subtilis + pGFP-rrnB

Today, the Metal Sensing team will assess the plates and judge whether the transformations have worked well.

Observations

No colonies grew on the LB alone plate or the LB Chloramphenicol plate with B. subtilis lacking pGFP-rrnB. However there was considerable growth on both of the LB + Chloramphenicol plates which contained pGFP-rrnB B. subtilis transformants - more colonies could be seen in the 200ul portion than on the 50ul portion but that is simply down to concentration of cells. Both transformations were a success, as can be seen in the above photograph.

Stochastic Switch Team

Goksel and Jess are all prepared for the Bacillus subtilis transformation procedure with these solutions made up

Today we are trying to transform Bacillus subtilis with pGFP-rrnB integration vector. Metal sensor team kindly inoculated B. subtilis cells into flask tubes and placed them into the shaking incubator. We labelled them as 1,2,3 and control.

We prepared four plates

• LB + Bsubtilis (+ Control, cells should grow on this plate)
• LB + CHL + B. subtilis (- control, cells will not grow on this plate)
• LB + CHL + B. subtilis + plasmid DNA(Cells should transform with the DNA)
• LB + CHL + B. subtilis + diluted plasmid DNA (Cells should transform with the DNA)

• 2xcompetence medium and 2xstravation medium were prepared.
• We used the 2nd and the 3rd tubes for the tests
• Incubated the samplesfor three hours at 37C
• Added the starvation medium and incubated for another 2 hours
• For each of the two cultures, added 0.4ml of the final solution from the culture and 10ul of DNA into an eppendorf tube.
• Placed the tubes in the shaking incubator for an hour. To do this we taped the tubes to the base of the incubator. N.B- Chris in the lab swapped the temperatures of the incubators, so we used the one that is opposite the -80 freezer. The starter overnight cultures are also in this incubator -these are labelled 1,2,3 control (we used 2 and 3).
• The cultures were plated out using glass beads and put in the 37 incubator.

Sporulation Tuning/Chassis Team

Today, we made our first attempt to recover the cwlD spores which were kindly sent to us by Anne Moir from Sheffield University as mentioned on the 4th of August.

Jane pipetting the cwlD spores out of the eppendorf tubes

We used the protocol for Method A as mentioned.

Preparation

The Lysozyme Stock Solution was made on the 7th of August.

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.

Note

Due to previous calculation errors, only 4ul of stock lysozyme was added to a ml of buffer solution. Instead, 40ml of stock lysozyme should have been added. As lysozyme is added to disrupt the bacterial cell wall, 4ul may not have been sufficient.

The experiment was performed again on Monday, 17th August. See Monday, 17th August for more details.

Protocol

In addition to the protocol for Method A, it is important to take note of the following points:

• 10ul of cwlD spores were added into the lysozyme and buffer solution.

The spores in the 37 degree incubator.

• 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 weight.
• The supernatant was removed from the eppendorf tube and the spores were resuspended in 1000ul of LB solution.

Note

The team faced difficulties in resuspending the spores.

The serial dilutions.

• A serial dilution was performed as illustrated below, and 50ul of each solution was plated out using glass beads on LB + Cm plates.
• In addition, 5ul of the cwlD spores were plated out using glass beads on LB + Cm plates. No colonies should grow on this plate as the spores were not treated.

Results

The following pictures shows the results for the recovery of the cwlD spores.

As mentioned, due to previous calculation errors, only 4ul of stock lysozyme was added to a ml of buffer solution in this experiment. A volume of 40ml of stock lysozyme should have been added instead. As lysozyme is added to disrupt the bacterial cell wall, 4ul may not have been sufficient, resulting in no or few colonies growing on the plates, as seen in the pictures below. The experiment was performed again on Monday, 17th August. See Monday, 17th August for more details.

In this plate, 5ul of spores were directly plated on without going through any treatment to see if any colonies grew.

cwlD mutant plate, without treatment.

The spores in this plate were not diluted, thus it is expected that the number of colonies growing in this plate would be greater than the other plates.

cwlD mutant plate, concentration = 1

Only one colony is seen growing on this plate, where a 1 time dilution was performed.

cwlD mutant plate, concentration = 10^-1

As can been seen in the following pictures, starting from the 2 times dilution onwards, no colonies can be seen growing.

cwlD mutant plate, concentration = 10^-2

cwlD mutant plate, concentration = 10^-3

cwlD mutant plate, concentration = 10^-4

cwlD mutant plate, concentration = 10^-5