MntH with amber stop codon with promoter for expression, no tRNA
This part encodes the manganese transporter, MntH, with one UAG stop codon in place of a leucine and a promoter for expression. MntH is involved with manganese homeostasis, a critical process for dealing with free radicals generated upon exposure to radiation that could potentially cause DNA damage.
Usage and Biology
Deinococcus radiodurans is a highly radiation-resistant bacterium that can survive after up to 12,000 Gy (absorbed radiation dose, Gray). The unique genes that allow it to better handle radiation exposure may provide ways to make E. coli and other organisms more resistant for applications of synthetic biology in tough environmental conditions, like space. Manganese is a cofactor for many of the radiation stress mechanisms in the cell, and so manganese transporters bring manganese into the cell during radiation stress. These transporters are particularly well-known in D. radiodurans, and expressing these proteins in E. coli has shown promise .
This part, as a part of the Amberless toolkit, should be used in Amberless E. coli. As it has one TAG stop, it will not express in other organisms. It will work in conjunction with the expression of the BBa_K1499251 supP tRNA part in amberless cells.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 215
Illegal NgoMIV site found at 1007
Illegal NgoMIV site found at 1080
- 1000COMPATIBLE WITH RFC
Verification of Part
The part was sequence verified in the pSB1C3 backbone before submission to the registry. Two reads, both in the forward direction, were obtained using VF2 and a primer that was designed to bind in the middle of the gene. The site-directed mutagenesis worked at our targeted TGA->TAG stop codon mutation.
The part has been tested alongside other radiation resistance genes (Figure 2). It does not confer resistance in DH5-alpha cells, as expected. It should only work in the Amberless chassis in conjunction with the BBa_K1499251 supP tRNA part.
1. Haiyan, S & Baoming, T (2010). Radioresistance analysis of Deinococcus radiodurans gene DR1709 in Escherichia coli.African Journal of Microbiol. Research, 4(13): 1412-1418.