Part:BBa_K730001
The mxaF gene is approximately 1.8 kb in size and encodes a 66-kDa polypeptide.
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.
Team Szeged_SA_RMG's contribution to this part: https://parts.igem.org/Part:BBa_K2418001 and https://parts.igem.org/Part:BBa_K2418002
Improved version of BBa_K730001 - 2
It was essential to find an appropriate promoter with high efficiency to enable the gene to work. Therefore, we looked for a promoter which can be found originally in Methylococcus capsulatus. The former iGEM team, iGEM12_juit found a nearly 1 000 base pairs long sequence which contained a promoter but they could not determine the exact location of the promoter. We did a search to find the 1 000 base pairs long sequence in Methylococcus capsulatus’ complete genome. This sequence contained not only the nucleotide sequence between two genes (the moxY and the mxaF) but did contain a partial part of each of the two genes. The putative moxY or mxaF used in this study did only contain the nucleotide sequence between the two genes, which must contain the promoter of either the moxY or the mxaF gene. Unfortunately, the orientation of the promoter is not known because the moxY and the mxaF genes are in different directions, therefore we could not determine neither the exact orientation of the promoter nor the exact sequence but we managed to approach the exact sequence of the promoter and apply it in such a way that the orientation was not needed to know.
The promoter was intended to ligate between the BglII restriction sites. This could enable the promoter to ligate in both orientations, randomly. The promoter was supplied with BglII restriction sites at both ends and addition AGTCAGTC nucleotides before and after each added AGTCAGTC nucleotides.
1. Addition AGTCAGTC bases
2. BglII restriction site
3. Promoter
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 120
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 120
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 120
Illegal BamHI site found at 816 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 120
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 120
Illegal NgoMIV site found at 447
Illegal NgoMIV site found at 585 - 1000COMPATIBLE WITH RFC[1000]
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