Function of Sulfite in E.coli

Sulfite reductase stable expressing cyanobacteria cost longer time and a lot of money to set up. To check the function of constructed gene in advance, we perform a chemical nephelometry assay to analyze the function. We follow a paper done by Prof. I. Lavilla (Microvolume turbidimetry for rapid and sensitive determination of the acid labile sulfide fraction in waters after headspace single-drop microextraction with in situ generation of volatile hydrogen sulfide, Analytica Chimica Acta 647 (2009) 112–116) to detect H2S. Due to our pTrc-kan-Cys I and pTrc-kan-Dsr gene can turn on in E.coli, we transform these gene into DH5 alpha E.coli and incubate in HSO3- contained LB medium. After 24hr and 48hr, we check the medium and detect H2S concentration.

Using Microvolume turbidimetry method can detect micro amount H2S

Instead of using complex machine and expensive HPLC, we use Zn(NH3)4·(OH)2 to absorp H2S, wich can form complex salt,〔Zn(OH3)6〕S. And then, add N,N–dimethy1-1,4-phenylenediammoniumdichloride(DPDA solution) and ferrite chloride to form methyl blue. Finally, by analyzing O.D.670, we can easily get H2S concentration. The reaction fomula are as followed:

〔Zn(OH3)6〕S+DADP/FeCl3→turbid/dark green-black(O.D.670nm)

(The same method used in Environmental Protection Administration Executive Yuan, Taiwan )

The picture shows DPDA structure and DPDA solution. When H2S exist, DPDA solution turn turbid

H2S serial dilution and standard curve detection

The curve is H2S standard serial diluted detected by O.D. 670nm