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Cloning of NosZ+NorCB gene:
Cloning of NosZ+NorCB gene:
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Ntrogen oxides are one of the mostnotorious pollutants in the modern days. At high temperature, nitrogen reactswith oxygen and form a binary compound of these two elements or a mixture ofsuch compound. These compounds are the main reason of smog and acid rain in theurban area; furthermore, they pose a major threat to the ozone in thestratosphere. The application of fertilizers and animal manure also result ingroundwater contamination by nitrate. Abnormally high nitrate concentrationcauses eutrophication and damages the environment. Anthropogenic disturbance ofNitrogen cycle accelerates the accumulation of nitrogen oxides, and suchsituation will deteriorate further if we aren’t devoted to solve the environmentalchallenge.<p></p>
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NosZ-NorCB
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NorCB is composed of two sub-unit – nitric oxide reductase subunit C and B. In fact, these two subunits are adjacent genes. And thus we cloned them together to produce a functional reductase.
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Recently, thesearch for biological nitrogen removal method from wastewaters and exhaust airhas come up with several promising methods; however, most of them just tookadvantage of some special bacteria combined with industrial procedures. On thecontrary, our iGEM project aim to reduce nitrogen oxides and oxidize sulfidecompounds at the same time. During the processes of denitrification, sulfidecompounds and nitrate act as electron donor and acceptor respectively. Thisreaction is also known as sulfide-driven denitrification. Researchers havereported that E. coli can perform such reaction when expresses sqr gene from R. capsulatus. Herein, we enable certaintype of cyanobacteria to take advantage of sulfide and reduce nitrogen oxidecompounds into nitrogen. The BLAST result shows that sqr genes are homolog in R. capsulatus and Synechocystis sp. PCC 6803. For denitrification, we plan to getaccess to Thiobacillus denitrificans,the well-known chemolithotrophic organisms. Nevertheless, we later found itdifficult to obtain the specific strain we need. According to NCBI database,enzymes for denitrification such as nir, nor, nos share great similaritybetween Thiobacillus denitrificans andPseudomonas aeruginosa PAO1, so weadopted P. aeruginosa PAO1 instead andexpressed the enzymes mentioned above in Synechocystissp. PCC 6803. These denitrifying enzymes are functional under aerobiccondition, yet like all cyanobacteria, Synechocystissp. PCC 6803 produces oxygen during photosynthesis. Fortunately, whensulfide presents in the environment and sqr is expressed, it will ceaseproducing oxygen and use sulfide as an electron donor for carbon dioxidephotoassimilation. Together with dsrI and dsrII enzymes from Desulfovibrio desulfuricans, ourengineered organisms are capable of reducing three major oxides pollution –nitrogen, sulfur and carbon oxides.<p></p>
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NosZ is the structure gene of nitrous reductase. Furthermore, it performs the last procedure of bio-denitrification in our model. Obviously, the reason we cloned NorCB and NosZ on the same plasmid is to connect the continuous denitrifying procedures together.
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In addition, both NorCB and NosZ are originated from'' Pseudomonas aeruginosa PAO1''.
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<p></p>
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Moreover, ourproject has been put into practice thanks to the cooperation with Chung HwaPulp Corporation. The wastewater generated by Pulp factories contains enormoussulfide compounds and nitrate, which bring the annoying odors as well as thecontamination to the local environment. Fortunately, our project seems to bethe solution to their problem. This also demonstrates the potential andpossibility of commercialized our project. On top of that, the engineeredcyanobacteria can become the third endosymbiosis organelles with the help ofdivision inhibitor, gene for invasion. After installing our designation intoplants or even human cells as artificial organelles, we grant eukaryotes theability to survive in extreme environments as horrible as Venus in case thespace immigration is necessary on day.<p></p>
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Latest revision as of 17:56, 25 September 2012
NosZ-NorCB
Cloning of NosZ+NorCB gene:
NosZ-NorCB
NorCB is composed of two sub-unit – nitric oxide reductase subunit C and B. In fact, these two subunits are adjacent genes. And thus we cloned them together to produce a functional reductase.
NosZ is the structure gene of nitrous reductase. Furthermore, it performs the last procedure of bio-denitrification in our model. Obviously, the reason we cloned NorCB and NosZ on the same plasmid is to connect the continuous denitrifying procedures together.
In addition, both NorCB and NosZ are originated from Pseudomonas aeruginosa PAO1.
We clone NosZ and NorCB and ligate them together. However, since there are PstI enzyme cutting site inside the NorCB, so we clone a new pSB1C3 backbone which contain EcoRI.XbaI.SpeI.SbfI enzyme cutting site.
Sequence and Features
Assembly Compatibility:
10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 1845 Illegal PstI site found at 2489 Illegal PstI site found at 3605
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 1845 Illegal PstI site found at 2489 Illegal PstI site found at 3605 Illegal NotI site found at 3764
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1517 Illegal BamHI site found at 2658
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 1845 Illegal PstI site found at 2489 Illegal PstI site found at 3605
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 1845 Illegal PstI site found at 2489 Illegal PstI site found at 3605 Illegal NgoMIV site found at 908 Illegal NgoMIV site found at 1072 Illegal NgoMIV site found at 2684 Illegal NgoMIV site found at 2726 Illegal NgoMIV site found at 3628 Illegal AgeI site found at 282 Illegal AgeI site found at 1381
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1638 Illegal BsaI.rc site found at 667 Illegal BsaI.rc site found at 1312 Illegal BsaI.rc site found at 1940
