Part:BBa_K2257006

hydrogen sulfide sensor sequence

SQR is a NAD(P)/FAD-dependent oxidoreductase, which oxidizes S2- to zero-valent S0. SqrR, encoded by sqrR, is a constitutively expressed suppressor protein of sqr promotor. RFP is the indicator. When there is no H2S in the environment, SqrR combines with the sqr promotor and keeps the expression of both the enzyme and RFP at a low level. While in the case where H2S exists, it will be oxidized by the small amount of expressed enzyme, and the product will interact with SqrR, forming a tri- or tetrasulfide cross-links linking C41 and C107 on the same subunit of SqrR. The change in the structure of SqrR makes it fall off from the sqr promotor and thus the expression of SQR and RFP turns on. The part can be used to detect hydrogen sulfide at a μmol level.

Design

As to the hydrogen sulfide sensor, we also designed a whole-cell biocatalytic system, displaying the concentration of hydrogen sulfide by the compound’s influence on specific genes’ expression in modified E.coli. Our idea is inspired from a type of bacterium that can live on sulfide.

Experiments

A plate sensitive assay measuring S2– tolerance of E. coli cells with constructed probe pathway. All plates were incubated at 37℃ for 18 h before being read. No significant influence appeared to the growth of E. coli at a concentration lower than 10mmol/L.

Figure 1.

We analysised the product by dual-enzyme digestion and electrophoresis.

Figure 2.Whole-cell sequence dual-enzyme digestion

RFP responsiveness of the detector system. Cells were grown to midlog phase under aerobic conditions and 0 ~ 250 μM Na2S. Cells were harvest after 17h and assayed for fluorescence intensity. Error bars indicate SD of the mean.

[[File:T-Nanjing-China-h2s-6.png|600px|thumb|center|a)][File:T-Nanjing-China-h2s-8.png|600px|thumb|center|b)][File:T-Nanjing-China-h2s-8.png|600px|thumb|center|c)]Figure 2.a)RFP responsiveness of the detector system.

           b) A visible photograph of a).

           c) Test of selectivity.]

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1373
  Illegal XhoI site found at 1574
  Illegal XhoI site found at 2261
  Illegal XhoI site found at 2375
  Illegal XhoI site found at 2573
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1304
  

The leading role in the system is a repressor protein called sqrR. Itinhibits the expression of Sqr via its binding to the promoter region of sqr. Sqr is a protein that oxidizes minus-two-valent S to zero-valent S.With the presence of hydrogen sulfide, the oxidized S can interact with the sqrR that is binding to the Psqr, forming a tetrasulfide bond and terminating the inhibition.
In our design, we use red fluorescent protein as the indicator.When hydrogen sulfide exits, the gene transcription is activated, and the bacteria turns red.