Team:Newcastle/Labwork/4 August 2009

=Formal Lab Session - 4th August 2009=

= Overview = 
 * Sporulation Tuning/Chassis Team - attempted to recover the cwlD spores using Method A but made error in calculating preparation solutions
 * Metal Sensor Team -prepared Bacillus subtilis cells for transformation tomorrow along with chemicals needed - transformations will involve BioBricks BBa_C0077 and BBa_C0076

Sporulation Tuning/Chassis Team
Prof. Anne Moir from Sheffield University kindly sent us spores, containing deactivated versions of the genes cwlD and sleB/cwlJ. These are genes that allow germination, which we plan to disable for use as our metal container.

The two eppendorf tubes containing the spores have been placed in a yellow box (labelled "iGEM Sporulation Tuning Team 04/08/09 JM/JH")in the fridge and are:
 * HC401 (FrpC2, cwlJ:: Kan, sleB-lacZ Ery R)
 * AM1877 (cwlD,Cmr, trp C2)

In the protocol given to us by Prof. Moir, there are two methods (Methods A and B), which can be used to recover the cwlD spores. However, neither give complete restoration of germination. Currently we have no method for restoration of the sleB/cwlJ spores. The same protocol is used to recover the sleB and cwlJ spores.

Referring to Prof. Moir's protocols, Method A is a fast method which results in partial germination of approximately 0.1%. Method B on the other hand, is a slow method which involves stripping of the spore coat layers for improved germination of approximately 10% recovery. Due to the lack of time, the group decided to go with Method A.

Preparation

 * Items needed:
 * Lysozyme (not available, ordered)
 * 10mM (0.01M) Potassium phosphate (Crystal form available, ordered)
 * 50mM (0.05M) KCl (Stock solution of 1M available)
 * 1mM (0.001M) MgCl2 (Stock solution of 0.5M available)
 * L-alanine (not available, ordered)

MW * Desired Volume (L) * Desired Molarity (M)
 * 0.01M Potassium Phosphate Solution:

174.18 * 0.1L * 0.01M = 0.174g

Therefore, 0.174g of Potassium Phosphate is mixed with 0.1L of DI water to make 0.1L of 0.01M Potassium Phosphate

1M (Stock Solution) / 0.05M (Desired Molarity) = 20
 * 0.05M KCl Solution:

Therefore, in order to obtain a solution of 0.05M, the stock solution needs to be diluted 20 times.

Desired volume = 0.1L

100ml / 20 = 5ml of KCl, and DI water would make up 95ml, producing 100ml of 0.05M KCl solution

0.5M (Stock Solution) / 0.001M (Desired Molarity) = 500
 * 0.001M MgCl2 Solution:

Therefore, in order to obtain a solution of 0.001M, the stock solution needs to be diluted 500 times.

Desired volume = 0.1L

100ml / 500 = 0.2ml of MgCl2, and DI water would make up 99.8ml, producing 100ml of 0.001M MgCl2 solution

The above 3 solutions have been prepared and sent for autoclaving. The group is now awaiting the ordered items, to carry on with the recovery of the cwlD spores. Meanwhile, we're in the progress of planning for the next lab session.

The above calculations are wrong, and the calculations have been redone, and experiment carried out on the 10th of August

Metal Sensor Team

 * Took notes of our things in the -20 freezer and -80 freezer in order to make a table on the wiki with all our stocks; see table details here
 * Innoculate LB with B.Subtilis transformed with BioBrick BBa_C0077 and BBa_C0076; 3 duplicates for each, stored in the shaking incubator overnight. These are for the Stochastic Switch Team.
 * Neil gave us the B.Subtilis transformation protocol from Prof.Colin Hardwood's lab
 * We scanned the protocol and sent it off by e-mail to everyone
 * We went to get the needed materials in Prof.Colin Hardwood's lab
 * Prepared B.Subtilis transformation material
 * 1 liter of SMM ( Spizizen Minimal Medium ), composed of 2g ammonium sulfate, 14g of dipotassium hydrogen phosphate, 6g potassium dihydrogen phosphate, 1g trisodium citrate, 0.2g magnesium sulphate.
 * 10ml of 40% glucose ( 4g of glucose + 10ml of distilled water )
 * We split the 1 liter SMM into 5 x 200ml and sent it for autoclaving
 * Neil gave us syringes and filters to sterilise the solution that can't be autoclaved.
 * Tomorrow we need to filtrate the 40% glucose as it can't be autoclaved and preapre the rest of the solutions.