Difference between revisions of "Part:BBa K1067006"

(→‎Characterization)
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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.
 
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
 +
 +
<p>A problem is that <b>N<sub>2</sub>O</b> couldn’t be detected precisely during the denitrification reaction, but we have discovered a new method.</p>
 +
          <img src="https://2020.igem.org/wiki/images/7/72/T--NAU-CHINA--cyccontribution3.png">
 +
          <p style="font-size: 12px !important;margin-top: 0px;text-align: center;">Fig.1. Schematic diagram of SBR device(Liu Guohua, et al.,<i>Environmental Engineering</i>. 2020)</p>
 +
          <p>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 N<sub>2</sub>O was determined by <b>Agilent 6890N gas chromatography (GC) equipped with ECD detector</b>.</p>
 +
          <p>The N<sub>2</sub>O 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 N<sub>2</sub>O occurs mainly in the aerobic stage, and its conversion rate is 2.84%.</p>
 +
          <img src="https://2020.igem.org/wiki/images/2/2a/T--NAU-CHINA--cyccontribution4.png">
 +
          <p style="font-size: 12px !important;margin-top: 0px;text-align: center;">Fig.2. N<sub>2</sub>O emission rates and N<sub>2</sub>O cumulative emission in A/O- SBR system(Liu Guohua, et al., <i>Environmental Engineering</i>. 2020)</p>
 +
          <p style="margin-bottom: 0">This study got some conclusions:</p>
 +
          <p style="margin: 0">①The release of N<sub>2</sub>O occured mainly in the aerobic stage of A/O-SBR sewage treatment pilot system.</p>
 +
          <p style="margin: 0">②The maximum release rate of N<sub>2</sub>O was 2.02 μgmin<sup>-1</sup> gmLSS<sup>-1</sup> 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.</p>
 +
          <p style="margin-top: 10px">Although the equipment is a little big to use in the laboratory, we can improve it in the future.</p>
 +
  
 
[[File:dtu-nir-char.png|600px|Nitrous oxide produced over time, measured with a microsensor in an anaerobic environment.  The times that nitrite was added is indicated by the vertical lines.  The data does not indicate that cells transformed with this biobrick (BBa_K1067006) show a strong response to nitrite.]]
 
[[File:dtu-nir-char.png|600px|Nitrous oxide produced over time, measured with a microsensor in an anaerobic environment.  The times that nitrite was added is indicated by the vertical lines.  The data does not indicate that cells transformed with this biobrick (BBa_K1067006) show a strong response to nitrite.]]

Revision as of 10:03, 27 October 2020

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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3445
    Illegal PstI site found at 6521
    Illegal PstI site found at 7162
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE 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
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3445
    Illegal PstI site found at 6521
    Illegal PstI site found at 7162
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE 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

A problem is that N2O couldn’t be detected precisely during the denitrification reaction, but we have discovered a new method.

         		<img src="T--NAU-CHINA--cyccontribution3.png">

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.

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%.

         		<img src="T--NAU-CHINA--cyccontribution4.png">

Fig.2. 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.


Nitrous oxide produced over time, measured with a microsensor in an anaerobic environment. The times that nitrite was added is indicated by the vertical lines. The data does not indicate that cells transformed with this biobrick (BBa_K1067006) show a strong response to nitrite.