Composite
Pc-pelB-rD

Part:BBa_K4225007

Designed by: Leon Ritchie Salim   Group: iGEM22_HKUST   (2022-10-08)
Revision as of 10:56, 10 October 2022 by Lrsalim (Talk | contribs) (→‎Contribution: HKUST 2022)

Pc-pelB-rDAO-6xHis

This is a composite part that consists of rDAO that is tagged with N-terminal pelB translocation tag (BBa_J32015) and induced by a constitutive promoter Pc (BBa_J23109). The presence of pelB allows the rDAO enzyme to be translocated into the periplasm of E.coli which allows better formation of disulfide bonds due to the presence of DsbA-DsbB and DsbC-DsbD systems. Note that the pelB translocation tag doesn’t influence the catalysis function of the rDAO, and thus rDAO will catalyze the conversion of diamine to H2O2 and other products. Teams that are planning to use bioamine as an input signal, such as histamine or cadaverine, may use this enzyme to convert the input to H2O2, which can activates many H2O2 inducible promoter such as oxySp(BBa_K4225001) and katGp(BBa_K4225004) with the assistance of OxyR protein(BBa_K1104200) as their transcription factor.

C-terminal 6xHis is added for characterization using western blotting.


Contribution: HKUST 2022

Summary

We analyzed the fluorescence intensity of GFP in an increasing concentration of H2O2. Since GFP is regulated under oxySp that responds to change of [H2O2] with the assistance of OxyR, we would expect a gradual increase of fluorescence with increasing concentration of H2O2.

Ninhydrin and Histamine Assay

Summary

We use ninhydrin and histamine to measure the activity of rDAO to decrease the concentration of histamine. From the experiment, we would expect a decrease of ninhydrin absorbance using pelB-rDAO compared to the negative control.

Experiment

pelB-rDAO (BBa K4225007) constructs are tested with 600 ppm of histamine alongside Pc-rDAO (BBa K4225021) as a positive control and pSB1C3 as a negative control. Constructs are inoculated in 5mL LB with chloramphenicol (CHL LB) for 16 hours. The constructs are then back diluted to 1.0 OD600. These steps are done to equalize the amount of cells for each culture. They are then added to 600 ppm of histamine and incubate for 1 hour. Before and after incubation for 1 hr, the samples are spun down. Afterwards, the supernatants are taken, mixed with ninhydrin and heated at 80oC for 20 min. Finally, the samples are placed in an ice box for 3 minutes and absorbance is measured at 570 nm.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1172
    Illegal BamHI site found at 2100
    Illegal BamHI site found at 2354
    Illegal XhoI site found at 970
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 156
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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