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From 2012.igem.org

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Engineered Vector

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Complete Description

Project Description

We are going to use the nif (From M.capsulatus) and nos (from Pseudomonas) genes. The mmo genes are present in to forms in M.capsualatus i.e sMMO(soluble MMO) and pMMO(particulate MMO). MMO enzyme catalyzes the conversion of methane to methanol. Once methanol is converted into formaldehyde it enters various biochemical cycles in the cell. Our system involves the utilization of the methanol as an inducer for MxaF promoter. The nosz gene is used to convert nitrous oxide into nitrogen. NosZ is a gene derived from P.aeruginosa and is used for nitrogen fixation. NifA is a specific transcriptional activator of the nif genes and acts in conjunction with RNA polymerase. Nif genes then converts the nitrogen into nitrate, which can be easily be taken up by the plant. The main reason for addition of fertilizer is to increase the inorganic nutrients in the soil. By utilizing NosZ and Nif genes we are converting the nitrous oxide into nitrate, thus reducing the need of fertilizers in the soil by increasing the nitrogen available to the plants. This is a small step towards a more environment friendly future.

Details:

MxaF:

Methylotrophic bacteria are a diverse group of microorganisms with the ability to utilize single-carbon (C1) substrates more reduced than carbon dioxide as their sole source of carbon and energy.Methanotrophs possesses native methanol-inducible promoters, notably promoters which are located upstream of genes that encode methanol dehydrogenase and other proteins required for its activity and enzymes required for the synthesis of the methanol dehydrogenase prosthetic group, pyrroloquinoline quinone. Of these, the promoter PmxaF has been thoroughly scrutinized both biochemically and in expression studies. In its native form in the chromosome, this strong promoter is methanol inducible. However, when this promoter is cloned in expression vectors, it acts essentially in a constitutive mode. The mxaF gene is approximately 1.8 kb in size and encodes a 66-kDa polypeptide. Our system involves the utilization of the methanol as an inducer for MxaF promoter. This would result in the diversion of the flux thus leading to a faster degradation of methane for the cell to survive.

NosZ:

The complete denitrification of nitrate by bacteria to dinitrogen (N,) is generally an anaerobic respiratory process. The last step involves the dissimilatory reduction of nitrous oxide (N,O), the free energy change of which can be coupled to phosphorylation (Zumft,1992; Zumft & Kroneck, 1990). nosZ is the structural gene for the periplasmic N,O reductase which is required to the conversion of nitrous oxide into free nitrogen. . The nitrous oxide that is emitted in the paddy fields is caused due to the excessive use of fertilizers. The microbes naturally present in the paddy field lead to the emission of nitrous oxide by utilizing these chemical fertilizers. . NosZ is a gene derived from P.aeruginosa and is used for denitrification.

NifA:

The biological nitrogen fixation reaction is catalyzed by a complex metalloenzyme called nitrogenase (6, 14). Nitrogenase is composed of two separately purified proteins, both of which are extremely oxygen sensitive. Expression of the nif genes is regulated at the transcriptional level by the products of nifA in response to molecular oxygen or ammonia. NifA is a specific transcriptional activator of the nif genes and acts in conjunction with RNA polymerase holoenzyme containing the alternative sigma factor, sigma 54. NifA binds to a characteristic palindromic motif, TGT-N10-ACA, also known as upstream activation sequence (UAS), that is located more than 100 bp upstream of nif promoters. The NifA protein has three arbitrarily designated domains (20): an amino-terminal domain which is implicated in regulatory function, a catalytic domain that interacts with the sigma-RNA polymerase holoenzyme, and a C-terminal helixturn-helix motif which recognizes the UAS on the nif promoters.

SacB:

Expression from sacB confers sensitivity to sucrose in a wide variety of gram positive and negative bacteria and thus has been used extensively for the last twenty years as a negative selectable marker. The Bacillus subtilis sacB gene encodes the enzyme levansucrase (EC 2.4.1.10), which is secreted by B. subtilis cells into the culture medium. Levansucrase is a transfructosylase catalyzing sucrose hydrolysis and levan synthesis. In addition, this enzyme is capable of adding fructosyl residues to a wide range of acceptor molecules. In Escherichia coli and other gram-negative bacteria such as Rhizobium, Agrobacterium, or Cyanobacterium species, expression of sacB is lethal in the presence of sucrose

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