Team:UCL London/From the lab/Expts

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Contents

Introduction

After the 4 stress detecting devices been constructed, our team conducted a series of experiments to test how they react to different stresses that can possibly occur during fermentation. The main focuses were on mis-folded protein, especially in periplasm, shear stress, and oxygen combustion levels of E.coli.


Promoter degP was thought to respond to mis-folded protein in the periplasm of E.coli. Various organic and inorganic chemicals which are suggested to cause protein denaturing in bacteria were used to induce protein mis-foldeding in the experiments. Important chemicals that were involved in our experiments are: Cu2+, Fe3+, NO3-, ethanol (C2H5OH), indole (C8H7N), 1-octanol (C8H18O) and toluene (C6H5CH3).


Promoter spy was thought to possibly detect high levels of shear stress during cultivation in a bioreactor. Additionally, spy showed response to some of the chemicals mentioned above as well.


NarK Promoter is switched on during anaerobic conditions (upregulated 100-fold and a further 8-fold in the presence of nitrate). Consequently, NarK was considered to detect low oxygen level, i.e. anaerobic metabolism. The difference between NarK promoter and mNarK promoter was tested by nitrate in the experiments illustrated below.

Copper 2+ ion & Ethanol Experiments

Aims

Procedures

  • In the morning inoculate Blank, DegP, Spy, RPU and TetR in 10mL LB. All except control with 100 micro g/L Kanamycin.
  • Prepare CuCl2 0.1Molar and Ethanol 1Molar.
  • From that add different concentrations to 96 well micro well plate plus 200 micro litre of culture to reach concentration of:
CuCl2 1mM 2mM 4mM 8mM 12mM 20mM 40mM
C2H5OH 10mM 20mM 40mM 80mM 120mM 200mM 400mM 600mM

*Plus one well without chemicals.

  • OD of cultures:
Blank degP+GFP spy+GFP RPU+GFP tetR+GFP
C2H5OH 0.09 0.63 0.92 1.15 0.12

Observation

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