Lab 208

Who was in the lab

Kristian

Procedure

Gel purification

Gel purified AMO with USER endings and Nir1 extraction fragment made in yesterdays PCR.

Lab 115

Main purpose

Run Experiment 2 anaerobically in order to characterize the behavior of E.coli.

Who was in the lab

Ariadni, Helen, Natalia

Procedure

Adjusting the temperature at 37 degrees and calibrating the probes as described in Appendix 5.

Following the protocol Experiment 2

Changing the steps :

6. 3 ml of the overnight culture (from glycerol stock) in 200 ml of medium with OD=0.0317.

7. Then added 3 ml more after 2 hours and the OD was 0.1046. The OD after 1 hour was 0.1340. Afterwards we added glycerol 1g in 10 ml of water and then added in our growing culture. Then after 40 min the OD was 0.2737. (Setup wavelength 600 nm).

12. The OD was measured 0.1324 instead of 0.3.

19. Add 0.5 ml of nitrite solution and continue by adding 0.5 ml after 10 minutes then we took 2.2 ml of sample, we added then 1 ml and we saw a slow response after 6 minutes. Afterwards when there was any change in the curve we spiked with 1 ml of nitrite. We continued as there was no response by adding 1 ml more and then by adding 2 ml more. We took 2 ml for sample in the end and another 2 ml for OD measurement where OD=0.1350. One hour after we took 2 ml of sample and then we spiked with 1 ml of NO₂. There was no response after 12 minutes. Then we spiked with 0.5 ml of N₂O and we show a big response.

Results

Colorimetric results

Ammonium

Measuring range 2-75 mg/L NH₄-N

start point- signal <2 mg/L

middle point- signal 2 mg/L

end point- signal <2 mg/L

Nitrate

Measuring range 1-25 mg/L NO₃-N

start point- signal <1 mg/L

middle point- signal <1 mg/L but visible pinker

end point- signal <1 mg/L but much visible pinker

Nitrite

Measuring range 0.02-1 mg/L NO₂-N

start point- signal <0.02 mg/L

middle point- signal 0.29 mg/L

end point- signal 0.70 mg/L after X2 dilution

Point before spiking with NO₂ at the end measurement 1.03 mg/L (x2 dilution)- 0.5 mg/L (x4 dilution)

N₂O measurement

Conclusion

The E.coli didn't grow as usual and the problem might be the minimal medium. Concerning the N₂O sensor, after adding for third time 1 ml of nitrite the response was slow and started to get stabilized after 6 minutes. When we continue adding more nitrite there was no other response.

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