Composite

Part:BBa_K3726038

Designed by: Santiago Barragán Ariza   Group: iGEM21_MADRID_UCM   (2021-10-19)


CDS_Lv0_MCGII

This composite part corresponds to a phytobrick compatible MoClo Lv.0 CDS element. This part is an artificial operon designed to express the enzymes ppc_CDS (BBa_K3726029), gcl_CDS (BBa_K3726031), CDS_mtkA (BBa_K3726033), CDS_mtkB (BBa_K3726035), CDS_mcl (BBa_K3726037).

Within this artificial operon each CDS element is preceded by an in-silico designed RBS sequence using DeNovo DNA software. Only the first coding sequence of the operon is not preceded by an RBS sequence which must be assembled during the level 1 MoClo reaction for the expression of the operon in a level 1 transcriptional unit.

This part has been used within the iGEM MADRID_UCM 2021 iGEM team for the expression of an artificial carbon fixation pathway.

The relative transcription initiation strengths of each RBS in the operon context has been calculated with DeNovo DNA Software. Results are depicted in the graphic below.

For MCGII

800px-T--MADRID_UCM--RBSMCGII.png

Since this part corresponds with the half part of a fully functional artificial carbon fixation pathway, relative expression of each enzyme required for optimal pathway functioning is controlled by their internal RBS.

On the one hand, Mcl and ppc enzymes are the most important enzymes of the MCG pathway. For this reason, codon optimization has been performed using DeNovo DNA Software, adjusting the codon usage for high expression in Synechococcus elongatus PCC7942. On the other hand, the rest of the enzymes that come from E.coli, though they have not been codon optimized, we designed a strong RBS to compensate for the expression levels.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1099
    Illegal EcoRI site found at 1540
    Illegal EcoRI site found at 3299
    Illegal EcoRI site found at 4415
    Illegal EcoRI site found at 4499
    Illegal EcoRI site found at 7554
    Illegal PstI site found at 1684
    Illegal PstI site found at 1783
    Illegal PstI site found at 2368
    Illegal PstI site found at 3326
    Illegal PstI site found at 3554
    Illegal PstI site found at 5064
    Illegal PstI site found at 6078
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1099
    Illegal EcoRI site found at 1540
    Illegal EcoRI site found at 3299
    Illegal EcoRI site found at 4415
    Illegal EcoRI site found at 4499
    Illegal EcoRI site found at 7554
    Illegal NheI site found at 4638
    Illegal PstI site found at 1684
    Illegal PstI site found at 1783
    Illegal PstI site found at 2368
    Illegal PstI site found at 3326
    Illegal PstI site found at 3554
    Illegal PstI site found at 5064
    Illegal PstI site found at 6078
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1099
    Illegal EcoRI site found at 1540
    Illegal EcoRI site found at 3299
    Illegal EcoRI site found at 4415
    Illegal EcoRI site found at 4499
    Illegal EcoRI site found at 7554
    Illegal BglII site found at 41
    Illegal BglII site found at 2081
    Illegal BglII site found at 3445
    Illegal BamHI site found at 2179
    Illegal BamHI site found at 2194
    Illegal BamHI site found at 5893
    Illegal BamHI site found at 6351
    Illegal BamHI site found at 7324
    Illegal XhoI site found at 871
    Illegal XhoI site found at 1516
    Illegal XhoI site found at 1990
    Illegal XhoI site found at 4520
    Illegal XhoI site found at 5187
    Illegal XhoI site found at 5320
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1099
    Illegal EcoRI site found at 1540
    Illegal EcoRI site found at 3299
    Illegal EcoRI site found at 4415
    Illegal EcoRI site found at 4499
    Illegal EcoRI site found at 7554
    Illegal PstI site found at 1684
    Illegal PstI site found at 1783
    Illegal PstI site found at 2368
    Illegal PstI site found at 3326
    Illegal PstI site found at 3554
    Illegal PstI site found at 5064
    Illegal PstI site found at 6078
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1099
    Illegal EcoRI site found at 1540
    Illegal EcoRI site found at 3299
    Illegal EcoRI site found at 4415
    Illegal EcoRI site found at 4499
    Illegal EcoRI site found at 7554
    Illegal PstI site found at 1684
    Illegal PstI site found at 1783
    Illegal PstI site found at 2368
    Illegal PstI site found at 3326
    Illegal PstI site found at 3554
    Illegal PstI site found at 5064
    Illegal PstI site found at 6078
    Illegal NgoMIV site found at 1627
    Illegal NgoMIV site found at 3016
    Illegal NgoMIV site found at 3190
    Illegal NgoMIV site found at 3340
    Illegal NgoMIV site found at 6514
    Illegal AgeI site found at 3641
  • 1000
    COMPATIBLE WITH RFC[1000]


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