Part:BBa_K1067006
Denitrification operon from Pseudomonas aeruginosa
This biobrick is the whole denitrification region from Pseudomonas aeruginosa PAO1 excluding only the enzyme in last step; Nitrous-oxide reductase ([http://ecocyc.org/PAER941193/NEW-IMAGE?type=LOCUS-POSITION&object=GLIQ-518&chromosome=CHROMOSOME-1 link to Biocyc]).
Usage and Biology
This brick has been shown hard to work with because it is a large region, all under one promoter. We purpose that splitting it into the 3 operons; like the conformation natively in P. aeruginosa and adding a separate promoter for each would make it easier to work with. If trying to to control the whole region under the same promoter one might experience premature mRNA transcripts because RNAP will fall of before the end of the region.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 3445
Illegal PstI site found at 6521
Illegal PstI site found at 7162 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3445
Illegal PstI site found at 6521
Illegal PstI site found at 7162
Illegal NotI site found at 4536
Illegal NotI site found at 6121 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3445
Illegal BglII site found at 197
Illegal BglII site found at 2942
Illegal BglII site found at 3335
Illegal XhoI site found at 6639 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 3445
Illegal PstI site found at 6521
Illegal PstI site found at 7162 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 3445
Illegal PstI site found at 6521
Illegal PstI site found at 7162
Illegal NgoMIV site found at 799
Illegal NgoMIV site found at 1926
Illegal NgoMIV site found at 1935
Illegal NgoMIV site found at 2357
Illegal NgoMIV site found at 2381
Illegal NgoMIV site found at 2467
Illegal NgoMIV site found at 3292
Illegal AgeI site found at 1663
Illegal AgeI site found at 6506 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 841
Illegal BsaI.rc site found at 973
Illegal BsaI.rc site found at 1706
Illegal BsaI.rc site found at 3391
Illegal BsaI.rc site found at 3693
Illegal BsaI.rc site found at 5125
Illegal BsaI.rc site found at 5676
Illegal SapI site found at 6182
Illegal SapI site found at 7434
Characterization
To characterize this biobrick, we ran an anaerobic experiment in which we added a known amount of nitrite to an anaerobic flask, and measured the nitrous oxide produced using a microsensor. We had a lot of trouble with the microsensor (see especially hour 4 on the below graph) and were not able to conclusively prove that we were producing nitrous oxide. We would recommend rerunning the experiment with a more reliable sensor.
Contribution
Group: NAU-CHINA iGEM 2020
The problem is that N2O couldn’t be detected precisely during the denitrification reaction, but we have discovered a new method.
Fig.1. Schematic diagram of SBR device(Liu Guohua, et al.,Environmental Engineering. 2020)
The gas samples are collected by the portable gas sampling pump every 30 min within one operating cycle and collected in a 1L gas sampling bag. The concentration of N2O was determined by Agilent 6890N gas chromatography (GC) equipped with ECD detector.
According to the changes of DO (Dissolved Oxygen), pH and ORP (Oxidation-Reduction Potential) in A/O-SBR biological denitrification system during one operating cycle, DO, ORP and pH have A good indicator effect on the process of nitrification and denitrification, and thus the accumulation of N2O--N can be judged and controlled.
Fig.2. Parameters change in the A/O- SBR system (Liu Guohua, et al., Environmental Engineering. 2020)
Here are the three equations to calculate the release rate, cumulative release and conversion rate of N2O during the operating period.
The N2O accumulation released in aerobic stage was 8.2 mg, about 6 times of that released in anoxic stage. According to the calculation, the conversion of N2O occurs mainly in the aerobic stage, and its conversion rate is 2.84%.
Fig.3. N2O emission rates and N2O cumulative emission in A/O- SBR system(Liu Guohua, et al., Environmental Engineering. 2020)
This study got some conclusions:
①The release of N2O occured mainly in the aerobic stage of A/O-SBR sewage treatment pilot system.
②The maximum release rate of N2O was 2.02 μgmin-1 gmLSS-1 during one operating cycle of the A/O-SBR small scale test system. The cumulative release rate reached 8.2 mg after 300 min of operation, and the accumulation of nitrite also reached the maximum value of 7.5 mg/L.
Although the equipment is a little big to use in the laboratory, we can improve it in the future.
//function/biosynthesis
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