Part:BBa_K1499205

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 [1].

This part, as a part of the [http://2014.igem.org/Team:StanfordBrownSpelman/Amberless_Hell_Cell 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 215
  Illegal NgoMIV site found at 1007
  Illegal NgoMIV site found at 1080
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

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.

Figure 1. Mutations were attempted in separate reactions at three different sites. This shows successful mutagenesis at the first site, with no mismatch (the mutation was written into the reference sequence).

Results

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.

Figure 2. Comparison of radiation resistance genes by measuring the fraction of colonies present after exposure to different amounts of UVC radiation. SOD was a resistance gene with unknown radiation resistance qualities. MntH Mut3 was a version of MntH with a UAG stop codon for leucine. Only uvsE-transformed cells showed an indication of radiation resistance.

References

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.