• 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 see how well they could live up to their designed function and also how they react to different stresses that could occur during fermentation. One focus of the experiments was chemicals believed to harm the cell envelope and cause aggregation mis-folded proteins in periplasm. Another focus of the testing was on responsiveness to very low levels of dissolved oxygen or anaerobic conditions.

The [http://partsregistry.org/wiki/index.php?title=Part:BBa_K239000 DegP promoter] is activated by the two-component system CpxAR and transcription is initiated by Sigma-E. It 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-folding 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).

The [http://partsregistry.org/wiki/index.php?title=Part:BBa_K239001 Spy promoter] is activated by the two-component system CpxAR as well as much the smaller two-component regulon BaeSR. It was thought to respond to similar stress as DegP but be less dependent on the growth phase of the organism. Spy also showed response to some of the chemicals mentioned above. We believe that both DegP and Spy have a high probability of being able to detect mechanical shear stress. But those experiments remain to be conducted.

The [http://partsregistry.org/wiki/index.php?title=Part:BBa_K239005 NarK promoter] is switched on during anaerobic conditions upon binding to the protein Fnr. The sequence also contains binding regions for the protein NarL and should therefore also be weakly activated in the presence of nitrate. The [http://partsregistry.org/wiki/index.php?title=Part:BBa_K239006 modified NarK promoter (mNarK)] was designed to have an optimised higher affinity for Fnr, stronger activity under anaerobic conditions and also to be less influenced by nitrate.

A Series of Growth Curve Experiments (OD&Fluorescent without stress)

Aims

1. Invesgating the general growth cruve of the host organism (E.coli W3110), with and without iGEM plasmids.
2. Measuring the fluorescent of the devices under normal condition, i.e. with no stresses at all.

Procedures

• Grow –ve control, RPU, degP+GFP device, spy+GFP device, NarK+GFP device and tetR+GFP device in 10ml LB overnight.
• Measure the OD of the overnight cultures.
• Determine the volume to inoculate in 50ml fresh LB with Kana in a conical flask, in order for all the culture starts to grow at small or similar OD.
• Take out samples to measure OD by 1 hour interval, for 6 to 8 hours(various from experiments date).
• Measure fluorescent levels after OD measurements.
• Take OD and fluorescent measurements after 12 hours of the last measurment mentioned above.

Observation

As shown in the Results.

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.

High OD E.coli culture with 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.

Copper optimisation Experiment

Aims

Procedures

• In the morning inoculate Control, DegP, Spy and RPU in 2mL LB. All except control with 100 µg/L Kanamycin.
• After OD measurement dilute to reach OD 0.15-0.17 for all measurements then apply 200 µL × 24 for each culture on a 96-well plate.
• On the 96-well plate: apply different CuCl2 concentrations ranging from 0-11.5 mM with an increase of 0.5 mM for each well.

Cu2+, Fe2+, Cl- Experiment Part I

Aims

Procedures

• Inoculate 30 µL of high concentrated cell culture from refrigerator (OD ca 10) control, DegP, Spy and RPU into 4 mL of LB. DegP, Spy and RPU containing 100 µg/L Kanamycin.
• Make CuSO4 0.1M, FeSO4 0.1M, NH4Cl 0.1M
• OD measurement

• Dilute into 6 mL to reach OD between 0.037 to 0.040.

(DegP, Spy and RPU LB containing 100 µg/L Kanamycin. )

• Add 200 µL of each culture into 24 wells on a 96-well plate.

Leave one well from each without any chemicals.

Add CuCl2 into 7 wells to reach concentrations of 2,3,4,5 and 10 mM.

Add CuSO4 into 8 wells to reach concentrations of 1,2,3,4,5 and 10 mM.

Add FeSO4 into 8 wells to reach concentrations of 1,2,3,4,5 and 10 mM.

Add NH4Cl into 8 wells to reach concentrations of 2,4,6,8,10 and 20 mM.

• Take first Tecan measurement, leave plate on shaking at 37 °C and take another 12 measurements until 20:30 and another measurement the next morning.

Observation

Too low starting OD to get significant values. Next day result shows that the cells seem to survive better in iron than in copper and to be unaffected by NH4Cl.

Cu2+, Fe2+, Cl- Experiment Part II

Procedures

• Repeating yesterday’s experiment but with higher OD.
• Inoculating 50 µL Control, 100 µL DegP, 100 µL Spy and 120 µL RPU (from high OD cultures from refrigerator) into 4 mL of LB media. DegP, Spy and RPU LB containing 100 µ g/L Kanamycin.
• OD measurement:

• Dilute into 6 mL of LB to reach OD 0.22 for all samples.(DegP, Spy and RPU LB containing 100 µ g/L Kanamycin.)
• Prepare the 96-well plate in the same way as yesterday from these samples with higher OD.
• Start Tecan measurements and take 10 measurements (roughly 30 minutes interval) until 19:00.

Observation

At 19:00 the fluorescence for the well with 4mM of CuCl2 has increased 60% for DegP and decreased 33% for RPU. The well with 5mM FeCl2 had also increased with 60% for DegP, possibly slightly for Spy but only decreased marginally for RPU.

Indole Experiment

Aims

Procedures

• Make 0.1 Molar Indole.
• Inoculate: Control, DegP, Spy and RPU from high cell density culture in refrigerator into 5 mL LB.

All except control with 100 micro g/L Kanamycin.

• OD of cultures:

• From 0.1M Indole and 0.1 M CuCl2 add different concentrations to 96 well micro well plate plus 200 µL of culture to reach concentration of:

• From take 5 fluorescent measurements within next 45 minutes with Tecan.

Observation

No results except that RPU fluorescent is decreasing immediately with higher CuCl2 concentrations and seems unaffected by Indole over the time span.

Indole (Nitrate) Experiment

Aims

Procedures

• Inoculate Control, DegP, Spy and RPU from glycerol stock into 5 mL LB. All except control with 100 µg/L Kanamycin.
• Dilute Indole 0.1M to 0.05M and keep it heated at ca 70 degrees Celsius to stay dissolved in the solution.
• At OD ca 1 for the cultures add 200 µL of each culture into 16 wells on a 96-well plate.
• Leave on well for each culture without any chemicals.
• Add 0.1M CuCl2 into 5 wells to reach concentrations of: 2mM, 3mM, 4mM, 5mM, 6mM.
• Add 98% ethanol into 6 wells to reach concentrations of: 3%, 4%, 5%, 6%, 7%, 8%.
• Add warm 0.05M Indole into 6 wells to reach concentrations of: 2mM, 4mM, 6mM, 8mM, 10mM, 12mM.
• Take 6 Tecan measurements between 21:45 and 22:35 while incubating the plate on 37°C, 300RPM.

Observation

No activity for these measurements but the next day (09:40-10:50) the fluorescence is high for Indole 6mM for both DegP and Spy.

Procedures II

• Same preparation as for the experiment above except that the bacteria are inoculated into 20 mL and then concentrated to an OD of 5-6. (Including adding 5% glycerol and letting the cultures readapt for 60 minutes)
• Take 6 Tecan measurements between 19:45 and 22:30 while incubating the plate on 37 degrees Celsius, 300RPM.

Observation II

No stress induced fluorescence could be detected this evening or the next morning but high fluorescent of DegP without stress already by start of the measurements.

Nitrate Experiment

Aims

Procedures

• Inoculate Control, NarK, mNarK and RPU from glycerol stock into 5 mL LB. All except control with 100 µg/L Kanamycin.
• At OD ca 1 for the cultures add 200 µL of each culture into 8 wells on a 96-well plate.
• Add 7 different volumes of 1M NaNO3 into 7 different wells for each culture to reach concentrations of: 40mM, 80mM, 120mM, 160mM, 200mM.
• Leave on well for each culture without any chemicals.
• Take 6 Tecan measurements between 21:45 and 22:35 and in the morning while incubating the plate on 37°C, 300RPM.

• Same preparation as for the experiment above except that the bacteria are inoculated into 20 mL and then concentrated to an OD of 5-6. (Including adding 5% glycerol and letting the cultures readapt for 60 minutes)

Take 6 Tecan measurements between 19:45 and 22:30 while incubating the plate on 37°C, 300RPM.

Oxygen Level Experiment(Nitrogen and sealed eppendorf tube test)

Aims

Procedures

• In the morning inoculate NarK and mNarK from glycerol stock into 2 mL of LB containing 100 µg/L Kanamycin.
• OD measurement:

NarK: 0.5, mNarK: 1.3.

• Inoculate 100 µL of NarK and 40 µL of mNarK respectively into 3 Falcon 50mL falcon tubes containing 5mL of LB+Kanamycin and 2 eppendorf 1.5mL containing 1.5 mL of LB+Kanamycin (the eppendorf tubes are completely full).
• Seal the eppendorfs and apply plastic film around the lids to make sure no gas can go in or out of the tubes. From the Falcon tubes let one have the lid loose and for the other two spurge nitrogen gas into the to get rid of all the oxygen in the tubes.
• Spurge nitrogen in a hood to avoid aerosols being released, also contain the tubes in a plastic bag that is also filled with nitrogen.
• Spurge nitrogen 3 times 15 seconds into each tube, between each time seal the tube, shake it and let it rest for 30 seconds.
• After the tube is sealed the last time apply plastic film to make sure no gas can go in or out of the tube.
• Incubate over night at 37°C.
• Take Tecan measurements the next morning.

Observation

High fluorescence for the samples with access to oxygen and no fluorescence for all samples without access to oxygen.

Organic Chemicals Experiment(Toluene, 1-octanol, n-octane)

Aims

Organic chemicals like toluene, octanol can possibly cause protein mis-folding in bacteria. DegP and spy promoters are expected to detect the presence of relevant unfolded proteins in the periplasmic space.

Procedures

• Inoculate 0.5ml overnight E.coli culture (blank E.coli/RPU/degP device/spy device) into 10ml fresh LB media with Kanamycin except for blank E.coli.
• Incubate for 2 hours to 3 hours until they reach mid-log phase, i.e. OD around 1.0 at wavelength 600nm.
• Add different amounts of toluene, 1-octanol, and n-octane onto a 96 well plate as stated in the table.1 below.
• Add 200µL of E.coli culture into corresponding well.
• Measure fluorescent level immediately
• Incubate at 37°C with 300rpm shaking.
• Measure fluorescent at 10-15mins intervals within 2 hours.

Observation