Difference between revisions of "Part:BBa K1067006"

(→‎Characterization)
(→‎Characterization)
Line 15: Line 15:
 
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.
  
−
[[File:dtu-nir-char.png|300px|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.]]
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 13:56, 12 October 2013

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.

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.