Lab 115

Main purpose

Investigate nitrite stability in anaerobic, untransformed E. coli

Who was in the lab

Helen, Kasia

Procedure

Following Experiment 1a in order to ensure that any produced nitrite from Mutant 1 will not be consumed by any of the pathways in native E. coli.

Results

Mg of Nitrogen (as Nitrite or Ammonia) per liter measured for varying concentrations of Nitrite added at t=0. All the experiments were performed anaerobically. Samples were measured every 20 min for 3 hours.

Conclusions and Discussion

Under anaerobic conditions, nitrite is consumed and converted to ammonium. Since we want to convert the nitrite to nitrous oxide, a functional prototype will require either that the pathway to nitrous oxide be designed to outcompete this pathway, or that this pathway that consumes nitrite be knocked out (and the transformants grown in a medium containing plentiful ammonium so they do not need to synthesize it).

Lab 208

Main purpose

• miniprep of TAT, Sec, and Nir for sequencing
• LB medium preparation (2L)
• Pick up new PAO1 cultures
• Make new colony PCR to get Nir from the newly acquired colonies.

Who was in the lab

Kristian, Julia

Procedure

PCRs:

• pZA21::RFP with primer pair 13 58C and 3:00.
• araBAD from K808000 with primer pair 12 61C and 1:00
• Colony PCR with first PAO1 colony but with a gradient of concentrations from 1x to 500x
• Colony PCR with the 4 new plates of PAO1, concentration x1

Results

See tomorrows gel.

Conclusion

Nothing to conclude from today's work.

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