Coding

Part:BBa_K2560262:Design

Designed by: Daniel Marchal   Group: iGEM18_Marburg   (2018-09-30)
Revision as of 21:53, 12 October 2018 by Marchal (Talk | contribs)


mcrN - N-terminal domain of Malonyl-CoA Reductase from Chloroflexus aurantiacus


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 885
    Illegal BamHI site found at 213
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1188
    Illegal AgeI site found at 1270
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The part contains the 5' 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_K2560261 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 1-549

Accession number of encoded protein: AAS20429

mcrN 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

Marburg Toolbox

We proudly present the Marburg Collection, a novel golden-gate-based toolbox containing various parts that are compatible with the PhytoBrick system and MoClo. Compared to other bacterial toolboxes, the Marburg Collection shines with superior flexibility. We overcame the rigid paradigm of plasmid construction - thinking in fixed backbone and insert categories - by achieving complete de novo assembly of plasmids.

36 connectors facilitate flexible cloning of multigene constructs and even allow for the inversion of individual transcription units. Additionally, our connectors function as insulators to avoid undesired crosstalk.

The Marburg Collection contains 123 parts in total, including:
inducible promoters, reporters, fluorescence and epitope tags, oris, resistance cassettes and genome engineering tools. To increase the value of the Marburg Collection, we additionally provide detailed experimental characterization for V. natriegens and a supportive software. We aspire availability of our toolbox for future iGEM teams to empower accelerated progression in their ambitious projects.


Figure 3: Hierarchical cloning is facilitated by subsequent Golden Gate reactions.
Basic building blocks like promoters or terminators are stored in level 0 plasmids. Parts from each category of our collection can be chosen to built level 1 plasmids harboring a single transcription unit. Up to five transcription units can be assembled into a level 2 plasmid.
Figure 4: Additional bases and fusion sites ensure correct spacing and allow tags.
Between some parts, additional base pairs were integrated to ensure correct spacing and to maintain the triplet code. We expanded our toolbox by providing N- and C- terminal tags by creating novel fusions and splitting the CDS and terminator part, respectively.


Parts of the Marburg Toolbox




Tags and Entry Vectors




  • K2560001 (Entry Vector with RFP dropout)
  • K2560002 (Entry Vector with GFP dropout)
  • K2560005 (Resistance Entry Vector with RFP Dropout)
  • K2560006 (Resistance Entry Vector with GFP Dropout)
  • K2560305 (gRNA Entry Vector with GFP Dropout)