Part:BBa_K896001

CysI (Sulfite reductase)

Our engingeered cyanobacteria can remove Sulfur Oxides (SOX, SO2),main precursors of air pollution and produce H2S for futher uses(Sulfide-Quinone Reductase,sqr).

CysI( surfur reductase) :This is a enzyme that can reduce (HSO3)- to H2S using the embedded electron transfer system in pseudomonas aeruginosa PAOI.

[http://www.rcsb.org/pdb/explore/explore.do?structureId=3MM5 sulfite reductase in complex with the substrate sulfite]

Usage and Biology

1. From clinical research, we fund out that Pseudomonas aeruginosa can produce great amount H2S.So, we logical assumed there are some pivotal enzymes can transfer (HSO3)- to H2S.

2. We searched for [http://www.genome.jp/kegg/ KEGG] and [http://www.ncbi.nlm.nih.gov/ NCBI] and found out CysI, a sulfite reductase inside [http://www.ncbi.nlm.nih.gov/protein/NP_250529.1 Pseudomonas aeruginosa PAOI].

3. We've already constructed CysI gene into E.coli and cyanobacteria and used bacteria to remove SO2 in our environment. Also, this bacteria produced H2S, which is a substrate for Sulfide-Quinone Reductase(sqr).

4.These potential CysI transformed bacteria can perform bioremediation, also are a powerful species for Veniusian!!

Gene construction & cloning

1.This gene is constructed by NYMU-Taipei 2012 iGEMers

2.we get the whole gene sequence of Cys I from NCBI web (http://www.ncbi.nlm.nih.gov/gene/878581).

3.Cys I contain endogenous EcoRI and PstI site, so we can’t clone into PSB1C3 directly.As a result, We create a noval cassette- new pSB1C3( Mfe I-Xba I -pSB1C3-Sbf I-Spe I) for easily cloning.

4.Taking advantage of MfeI and EcoRI are compatible, also SbfI and XbaI, we can easily clone Cys I gene into pSB1C3 standard biobrick.

5.Enzyme check by XbaI and SbfI, we can get ~1700bp band, which means our gene constraction is correct!!

A powerful cassette for CysI engingeering, you can use customerized promoter as you want!!

Novel H2S detection assay

1.To check the function of constructed gene in advance, we perform a chemical nephelometry assay to analyze the function.

2.We follow a paper: [http://www.ncbi.nlm.nih.gov/pubmed/19576394 Lavilla I,Pena-Pereira F et al, 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.

3.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.

4.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.

5.The same method used in Environmental Protection Administration Executive Yuan, Taiwan: [http://www.niea.gov.tw/niea/AIR/A40671A.htm 排放管道中硫化氫檢驗法－甲烯藍比色法]

The reaction fomula and the compound of DPDA

Function assay of CysI

1.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.

2.Our CysI transformed E.coli can produce much more H2S, compared to the blank control Kan E.coli

The standard curve and regression line of H2S standard product(left). The data shows that CysI Transformed E.coli produced more H2S.

Practical application of CysI

1.To further practical use, we believe our artificial creatures have high ability to convert sulfite from industrial waste and keep our environment clean.

2. By Co-culture with our Cys I expressing E.coli for 24hr and 48 hr, we found out that the concentration of H2S increase dramatically,which means sulfite decreased!! Both gas phase and water phase of each group are analysed by Microvolume turbidimetry method (mentioned above).

Using our powerful CysI bacteria, It's a efficient way to remove industrial wastes!!

Other related part for Sulfur Oxide Terminator

1.SQR(sulfide quinone reductase):BBa_K896000

2.Dsr(sulfide quinone reductase):BBa_K896002

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 655
  Illegal PstI site found at 121
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 655
  Illegal PstI site found at 121
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 655
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 655
  Illegal PstI site found at 121
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 655
  Illegal PstI site found at 121
  Illegal NgoMIV site found at 394
  Illegal NgoMIV site found at 580
  Illegal NgoMIV site found at 722
  Illegal AgeI site found at 94
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 130
  Illegal BsaI site found at 1452
  

Details are in Design Notes!!

Functional Parameters

kegg	Pseudomonas aeruginosa PAO1: PA1838