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Denitrifying machine

Abstract Background

N itrogen oxides are one of the most notorious pollutants in the modern days. Anthropogenic disturbance of Nitrogen cycle accelerates the accumulation of nitrogen oxides, and such situation will deteriorate further if we aren’t devoted to solve the environmental challenge.

Methods Recently, the search for biological nitrogen removal method from wastewaters and exhaust air has come up with several promising methods; however, most of them just took advantage of some special bacteria combined with industrial procedures. On the contrary, our iGEM project aim to reduce nitrogen oxides and oxidize sulfide compounds at the same time. During the processes of denitrification, sulfide compounds and nitrate act as electron donor and acceptor respectively. This reaction is also known as sulfide-driven denitrification. Researchers have reported that E. coli can perform such reaction when expresses sqr gene from R. capsulatus. Herein, we enable certain type of cyanobacteria to take advantage of sulfide and reduce nitrogen oxide compounds into nitrogen. The BLAST result shows that sqr genes are homolog in R. capsulatus and Synechocystis sp. PCC 6803. For denitrification, we plan to get access to Thiobacillus denitrificans, the well-known chemolithotrophic organisms. Nevertheless, we later found it difficult to obtain the specific strain we need. According to NCBI database, enzymes for denitrification such as nir, nor, nos share great similarity between Thiobacillus denitrificans and Pseudomonas aeruginosa PAO1, so we adopted P. aeruginosa PAO1 instead and expressed the enzymes mentioned above in Synechocystis sp. PCC 6803. These denitrifying enzymes are functional under aerobic condition, yet like all cyanobacteria, Synechocystis sp. PCC 6803 produces oxygen during photosynthesis. Fortunately, when sulfide presents in the environment and sqr is expressed, it will cease producing oxygen and use sulfide as an electron donor for carbon dioxide photoassimilation. Together with dsrI and dsrII enzymes from Desulfovibrio desulfuricans, our engineered organisms are capable of reducing three major oxides pollution – nitrogen, sulfur and carbon oxides.


Bio-Bricks Part:BBa_K896004 NosZ-NorCB

In Pseudomonas aeruginosa PAO1, NorCB is found as two sub-units – nitric oxide reductase subunit C and B. As a matter of 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. Very few microorganisms process similar enzyme, and many of them are homologs according to NCBI database. Furthermore, it performs the last procedure of bio-denitrification – nitrous oxides to nitrogen – in our model. Obviously, the reason we cloned NorCB and NosZ on the same plasmid is to connect the continuous denitrifying procedures together.

Part:BBa_K896006 NirN-NirS

Cloning of NirN+NirS gene:Nitrite reductase is composed of several different subunits. Together, it can reduce nitrite to nitric oxide. We clone all these subunits together on the same plasmid in order to grant cyanobacteria a functional protein.

Part:BBa_K896007 Nap( Nitrate->Nitrite reductase)

Nap is periplasmic nitrate reductase, which is known for the ability to reduce nitrate into nitrite. In microorganisms, similar reductases are often observed. For instance, even E. coli has its own nitrate reductase and can use nitrate as final electron acceptor. Moreover, we acquire the Nap gene from Desulfovibrio desulfuricans ATCC 27774.

Since Nir and Nap complex together would link the reducing reaction from Nitrate to nitric oxide, we ligated these two gene together and expressed them on Synechococcus elongatus PCC 7942.

Practical Application Moreover, our project has been put into practice thanks to the cooperation with Chung Hwa Pulp Corporation. The wastewater generated by Pulp factories contains enormous sulfide compounds and nitrate, which bring the annoying odors as well as the contamination to the local environment. Fortunately, our project seems to be the solution to their problem. This also demonstrates the potential and possibility of commercialized our project. On top of that, the engineered cyanobacteria can become the third endosymbiosis organelles with the help of division inhibitor, gene for invasion. After installing our designation into plants or even human cells as artificial organelles, we grant eukaryotes the ability to survive in extreme environments as horrible as Venus in case the space immigration is necessary on day.


Sulfur-driven autotrophic denitrification: diversity ,biochemistry, and engineering applications (1 September 2010) by Ming-Fei Shao & Tong Zhang & Herbert Han-Ping Fang

Characterization of the membranous denitrification enzymes nitritereductase (cytochrome cd1) and copper-containing nitrous oxidereductase from Thiobacillus denitrificans (30 October 1995) by Ursula H. Hole • Kai-Uwe Vollack • Walter G. Zumft •Effi Eisenmann • Roman A. Siddiqui •Bärbel Friedrich • Peter M. H. Kroneck

Autotrophic denitrification for combined hydrogen sulfideremoval from biogas and post-denitrification by R. Kleerebezem and R. Mendez (2002)

Nitrous oxide production and consumption: regulation of gene expression by gas-sensitive transcription factors (7 July 2012) by Stephen Spiro

Mechanism of Sulfide-Quinone Reductase Investigated Using Site-Directed Mutagenesis and Sulfur Analysis (6 August 2002)by Christoph Griesbeck, Michael Schu¨tz, Thomas Scho¨dl, Stephan Bathe, Lydia Nausch, Nicola Mederer, Martin Vielreicher, and Gu¨nter Hauska