• Home
• Team
• Project
• Parts
• Modelling
• Sponsors
• Lab Book

Project

• Overview
• Choices Rationale
• Characterisation
• Safety
• Judging Comments

Sub-projects

• Metal Intake/Efflux
• Metal Sensing
• Sporulation Tuning
• Population Dynamics
• Stochastic Switch
• Metal Sequester
• Chassis
• Promoter Library

Wet Lab

• Lab Book
• Protocols

Meetings

• Meetings Calendar

Planning

• Timeline
• Ideas

Links

• Helping other teams
• Ethics
• T-Shirts
• iGEM Meetup
• Our City
• Fun and Games
• Sponsors
• Useful Links
• Contact Us
• Press Coverage

Lab Session 04/08/2009

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,Cm r, 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.

[Attach Prof. Moir's protocols]

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)

0.01M Potassium Phosphate Solution

MW * Desired Volume (L) * Desired Molarity (M) 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

0.05M KCl Solution

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

0.001M MgCl2 Solution

0.5M (Stock Solution) / 0.001M (Desired Molarity) = 500 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.