Coding

Part:BBa_K2560261:Design

Designed by: Daniel Marchal   Group: iGEM18_Marburg   (2018-09-30)
Revision as of 14:20, 30 September 2018 by Marchal (Talk | contribs) (→‎Design Notes)


mcrC - C-terminal domain of Malonyl-CoA Reductase from Chloroflexus aurantiacus


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 288
    Illegal NheI site found at 1690
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 361
    Illegal NgoMIV site found at 1975
    Illegal AgeI site found at 1524
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The part contains the 3' part of the mcr gene of C. aurantiacus (AY530019). This region is flanked by overhangs which are Phytobrick- and MoClo-compatible and by two BsaI recognition sites (Weber et al., 2011).

It was shown that a splitted version of Mcr with separated C- and N-terminal domains increases enzyme activity (Liu et al., 2013). If these splitted genes shall be used take BBa_K2560262 and this part. If the gene for the whole enzyme shall be used take BBa_K2560260

GGTCTCGAATG-coding_region-GCTTTGAGACC

The sequence was codonoptimized for V. natriegens ATCC 14048. According to Liu et al, we designed the part so that McrN contains amino acid 1-549 of the overall protein and McrC amino acid 550-1219 (Liu et al., 2013).

Source

Source of the part:

Genome: Chloroflexus aurantiacus strain OK-70-fl

Accession number of gene: AY530019, amino acid 550-1219

Accession number of encoded protein: AAS20429

mcrC was codonoptimized for V. natriegens and then synthetisized and integrated into the vector BBa_K2560002 via BsmBI

References

Liu, C., Wang, Q., Xian, M., Ding, Y., Zhao, G., 2013. Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement. PLoS One 8, 1–8. https://doi.org/10.1371/journal.pone.0075554

Strauss, G., Fuchs, G., 1993. Enzymes of a novel autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus, the 3-hydroxypropionate cycle. Eur. J. Biochem. 215, 633–43.

Weber, E., Engler, C., Gruetzner, R., Werner, S., Marillonnet, S., 2011. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS One 6, e16765. https://doi.org/10.1371/journal.pone.0016765