Team:Newcastle/Labwork/17 August 2009

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=Lab 17/08/09=
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<u>==Metal Sensor Team==</u>
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===Introduction and Summary===
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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.
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===Changes to protocol===
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There were no changes to the protocol described [https://2009.igem.org/Team:Newcastle/Project/Labwork/OurProtocols/Making_Agar_Plates#Making_up_starch_plates here] except for the fact that the chosen antibiotic is chloramphenicol
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===Work we did===
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* Using the [https://2009.igem.org/Team:Newcastle/Project/Labwork/OurProtocols/Making_Agar_Plates#Making_up_starch_plates '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.
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* 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.
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Revision as of 03:10, 16 August 2009

Contents

Lab 17/08/09

==Metal Sensor Team==

Introduction and Summary

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

  • 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.



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