Difference between revisions of "Part:BBa K896002"

(→‎Usage and Biology)
(→‎function assay & practical application of Dsr)
 
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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.
 
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, Desulfovibrio desulfuricans ATCC27774 and cloned its sulfite reductase, Dsr.
+
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.
 
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.
Line 27: Line 27:
 
5.These potential Dsr transformed bacteria can perform bioremediation, also are a powerful species for Veniusian!!
 
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”.
 +
 +
[[Image:R3.jpg]]
 +
 +
''' Membrane electronic prediction shows that no transmembrane domain exist'''
 +
 +
===gene construction & cloning===
 +
 +
1.This gene is constructed by [https://igem.org/Team.cgi 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!!
 +
[[Image:SSN4.jpg|600px]]
 +
 +
[[Image:R5.jpg]]
 +
 +
'''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: [https://parts.igem.org/Part:BBa_K896001#Novel_H2S_detection_assay CysI sulfite reductase:BBa K896001]
 +
 +
[[Image:R6.jpg|800px]]
 +
 +
[[Image:R7.jpg]]
 +
 +
'''Using chemical turbidimetry assay can detect H2S efficiently!! '''''
 +
 +
===function assay & practical application of Dsr===
 +
 +
1.To test the function and practical use for our society, we co-culture industrial wastes with our Dsr and Cys I expressing E.coli for 24hr and 48 hr. See [https://parts.igem.org/Part:BBa_K896001#Novel_H2S_detection_assay BBa K896001 :CysI] part
 +
 +
2.We found out that the concentration of H2S increase dramatically in CysI and Dsr transformed E.coli, compared to the blank control Kan transformed E.coli.That is to say, we clean industrial waste sulfite successfully!!Both gas phase and water phase of each group are analysed by Microvolume turbidimetry method (mentioned above).
 +
 +
[[Image:R8.jpg]]
 +
 +
'''By simplily co-culture the waste gas and water with our modified cyanobacteria and E.coli, we can clean the pollution!!'''
 +
 +
[[Image:SSN2.jpg|600px]]
 +
 +
 +
 +
 +
[[Image:SSN3.jpg|600px|]]
 +
 +
'''Using our powerful Dsr and CysI bacteria, It's a efficient way to remove industrial wastes!!(cmsumeing sulfite and form H2S)'''
 +
 +
===Other related part for Sulfur Oxide Terminator===
 +
 +
1.[https://parts.igem.org/Part:BBa_K896000 SQR(sulfide quinone reductase)]:BBa_K896000
 +
 +
2.[https://parts.igem.org/Part:BBa_K896001 CysI (Sulfite reductase)]]:BBa_K896001
 +
  
  

Latest revision as of 07:40, 29 September 2012

Dsr (sulfite reductase)

borderless

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

DsrI.jpg

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

DsrII.jpg

[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”.

R3.jpg

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!! SSN4.jpg

R5.jpg

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

R6.jpg

R7.jpg

Using chemical turbidimetry assay can detect H2S efficiently!!

function assay & practical application of Dsr

1.To test the function and practical use for our society, we co-culture industrial wastes with our Dsr and Cys I expressing E.coli for 24hr and 48 hr. See BBa K896001 :CysI part

2.We found out that the concentration of H2S increase dramatically in CysI and Dsr transformed E.coli, compared to the blank control Kan transformed E.coli.That is to say, we clean industrial waste sulfite successfully!!Both gas phase and water phase of each group are analysed by Microvolume turbidimetry method (mentioned above).

R8.jpg

By simplily co-culture the waste gas and water with our modified cyanobacteria and E.coli, we can clean the pollution!!

SSN2.jpg



SSN3.jpg

Using our powerful Dsr and CysI bacteria, It's a efficient way to remove industrial wastes!!(cmsumeing sulfite and form H2S)

Other related part for Sulfur Oxide Terminator

1.SQR(sulfide quinone reductase):BBa_K896000

2.CysI (Sulfite reductase)]:BBa_K896001





Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1636
    Illegal EcoRI site found at 4648
    Illegal PstI site found at 826
    Illegal PstI site found at 3838
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1636
    Illegal EcoRI site found at 4648
    Illegal PstI site found at 826
    Illegal PstI site found at 3838
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1636
    Illegal EcoRI site found at 4648
    Illegal BglII site found at 1091
    Illegal BglII site found at 4103
    Illegal BamHI site found at 3007
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1636
    Illegal EcoRI site found at 4648
    Illegal PstI site found at 826
    Illegal PstI site found at 3838
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1636
    Illegal EcoRI site found at 4648
    Illegal PstI site found at 826
    Illegal PstI site found at 3838
    Illegal NgoMIV site found at 1741
    Illegal NgoMIV site found at 4753
    Illegal AgeI site found at 406
    Illegal AgeI site found at 1261
    Illegal AgeI site found at 3418
    Illegal AgeI site found at 4273
  • 1000
    COMPATIBLE WITH RFC[1000]

Details are in Design Notes!!

Functional Parameters

keggDesulfovibrio desulfuricans ATCC 27774