• Protocols
• _{Parts Used in the Project}
• Parts Submitted
• Experiments
• Results
• QC Infomation
• Further Experiments

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.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K239000 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).

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K239001 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.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K239005 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:

*Plus one well without chemicals.

• OD of cultures:

Observation

• Immediate result is that RPU loses fluorescent with increasing CuCl2 and loses all florescent for CuCl2 concentrations of 20mM and above and also gain florescent for all Ethanol concentrations with the highest florescent at 600mM (which increases 100%). No other significant result within 1 hour.

• Next morning, RPU has lost all florescent for CuCl2 concentrations of 8mM or above.
• DegP has increased it’s fluorescent by at most 30% for 4mM CuCl2. Spy is silent without stress and induced for CuCl2 concentrations 2-8mM with highest at 4mM.
• All are showing higher values for Ethanol.

Copper 2+ ion & Ethanol Experiments

Aims

Procedures

• From overnight culture of 2mL Control, DegP, Spy and RPU inoculate into 250 mL shake flasks containing 50 mL of LB. All except control with 100 µg/L Kanamycin.
• Concentrate by adding 2 mL of each culture into 20× 2 mL eppendorf tubes. Spin down at 6000 RPM for 5 min. Discard the media, re-suspend the pellets in 400 micro litre fresh LB and transfer into 50 mL falcon tube. Add 5 % glycerol and incubate for 90 min at 37°C, 300 RPM.
• OD measurement:

• Transfer 1800 µL of each culture into 4 Falcon tubes 15 mL.
• Add 230 µL of 98% ethanol to one of each culture’s 4 tubes to reach 2.5 M Ethanol concentration.

Add 46 µL of 98% ethanol to the second of each culture’s 4 tubes to reach 0.5 M Ethanol concentration.

Add 36 µL of 0.1 M Copper Chloride to the third of each culture’s 4 tubes to reach 2 mM CuCl2 concentration.

Add no chemicals to the fourth tube.

• Add 230 µL of 98% ethanol to one of each culture’s 4 tubes to reach 2.5 M Ethanol concentration.

Add 46 µL of 98% ethanol to the second of each culture’s 4 tubes to reach 0.5 M Ethanol concentration. Add 36 µL of 0.1 M Copper Chloride to the third of each culture’s 4 tubes to reach 2 mM CuCl2 concentration. Add no chemicals to the fourth tube.