Difference between revisions of "Part:BBa K896002"

Revision as of 00:20, 27 September 2012

Dsr (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).

[http://www.rcsb.org/pdb/explore/explore.do?structureId=3OR1 Crystal structure of dissimilatory sulfite reductase I (DsrI)]

[http://www.rcsb.org/pdb/explore/explore.do?structureId=3OR2 Crystal structure of dissimilatory sulfite reductase II (DsrII)]

Usage and Biology

1. By paper surveying, we found out a kind of anaerobe bacteria, sulfur reducing bacteria(SRB), which have powerful ability to reduce (HSO3)- to H2S.

2. We discussed eith several specialists and searched for [http://www.genome.jp/kegg/ KEGG] and [http://tw.search.yahoo.com/r/_ylt=A8tUwZckY2NQmwcAA.hr1gt.;_ylu=X3oDMTBydTdmYjgyBHNlYwNzcgRwb3MDMQRjb2xvA3R3MQR2dGlkAw--/SIG=11g0agsiu/EXP=1348719524/**http%3a//www.ncbi.nlm.nih.gov/ NCBI]. Finally we engingeered a common SRB, [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=27774&Template=bacteria Desulfovibrio desulfuricans ATCC27774] and cloned its sulfite reductase, Dsr.

3.There are two sulfite reductase operon, DsrI and DsrII, inside Desulfovibrio desulfuricans. They might be membrane form Dsr operon and free form Dsr operon.

4. We've already constructed Dsr 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).

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

Transmembrane domain prediction analysis

1.Sulfite reductases have two types,membrane form(located on cell membrane) and free form(located inside cell membrane).Among which, free form are the best choice since the folding and structure are more stable.

2.We use bioinformation tools- ExPASy(http://expasy.org/) to predict the transmembrane domain of each sulfite reductase. Because DsrI and DsrII are free form, we combine DsrI and DsrII gene to form “Dsr”.

Membrane electronic prediction shows that no transmembrane domain exist

gene construction & cloning

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

2.DsrI and DsrII also contain endogenous EcoRI and PstI site, so we clone them into noval cassette- new pSB1C3( Mfe I-Xba I -pSB1C3-Sbf I-Spe I).

3.We create a BamH I site inside of DsrI and DsrII, in order to combine them into a whole part, “Dsr”(two operon in one plasmid, hope to get higer rate ).

4.Enzyme check by XbaI and Spe I , we can get ~5800 bp, which means our gene constraction is correct!!

A powerful cassette for Dsr 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.The mechanism and procedure please follow: CysI sulfite reductase:BBa K896001

Using chemical turbidimetry assay can detect H2S efficiently!! '

Sequence and Features

Details are in Design Notes!!

Functional Parameters