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Lab 14/08/09

Metal Sensor Team

Introduction and Summary

Arun preparing LB + agar powder

So far we have shown that our Bacillus subtilis 168 cultures, once the GFP-rrnb DNA has been added, can survive on LB + Chloramphenicol media. This suggests that we have successfully transformed our B. subtilis cells as the wild type B. subtilis cells do not have this resistance. However we need to prove that the resistance inherited by the Bacillus subtilis bacteria is due to the GFP-rrnb and not acquired by other means. Today, we intend to pour some LB + Chloramphenicol + starch plates and then plate our 'transformed' Bacillus cells onto these plates. The reason for adding starch to the plates: the GFP-rrnb plasmid removes the Bacillus subtilis bacteria's ability to break down starch using the amyE enzyme - starch plates will prove the bacteria have been transformed.

Changes to protocol

There were no changes to the protocol described here except for the fact that the chosen antibiotic is chloramphenicol

Work we did

Two 1 litre flasks - one containing 500ml LB + agar with starch also present and the other flask containing 500ml of deionised water

Mathew pouring the LB starch + chloramphenicol plates under aseptic technique

• Using the 'pouring plates' protocol, we made up 1 litre of LB + agar + starch solution and once it had been autoclaved and properly prepared (with chloramphenicol - this is the resistance displayed in GFP-rrnB), the solution was poured into plates.

• These plates were then left on the bench for 1 hour. Three plates were taken from the stack and placed in the 42ºC incubator to dry off.

• Once the plates had dried, a grid of 46 squares was drawn onto the base of the plate. These squares were then numbered from 1-46.

• Within square 1, a mark was made in the agar surface (in the shape of an 'X')with wild type Bacillus subtilis.

• Within squares 2-46, marks were also made on the agar surface but this time it was with our transformed Bacillus subtilis (which should have taken up gfp-rrnb)

• The plate was then left in the 37ºC incubator overnight.

Chassis team

The test transformation 50ul plate.

The test transformation 200ul plate.

The test transformation control plate.

The test transformation water plate.

The cwlD plate.

The cwlD 10^-1 plate.

The cwlD 10^-2 plate.

The cwlD 10^-3 plate.

The cwlD 10^-4 plate.

The cwlD 10^-5 plate.

The no treatment cwlD plate.