Composite

Part:BBa_K3726028:Design

Designed by: Santiago Barrag�n Ariza   Group: iGEM21_MADRID_UCM   (2021-10-19)

CDS_Lv0_MCG-PK


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 3079
    Illegal PstI site found at 3708
    Illegal PstI site found at 4205
    Illegal PstI site found at 4804
    Illegal PstI site found at 5742
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 3079
    Illegal PstI site found at 3708
    Illegal PstI site found at 4205
    Illegal PstI site found at 4804
    Illegal PstI site found at 5742
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1618
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3079
    Illegal PstI site found at 3708
    Illegal PstI site found at 4205
    Illegal PstI site found at 4804
    Illegal PstI site found at 5742
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3079
    Illegal PstI site found at 3708
    Illegal PstI site found at 4205
    Illegal PstI site found at 4804
    Illegal PstI site found at 5742
    Illegal NgoMIV site found at 1195
    Illegal NgoMIV site found at 3304
    Illegal NgoMIV site found at 3764
    Illegal AgeI site found at 5317
    Illegal AgeI site found at 5400
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

In the upstream region the bases taatc corresponds with 5bp a spacer sequence to improve ribosomal translation, while AATG overhang creates an start codon that forces translation initiation of the downstream coding sequence.

In the downstream region, the extra “aa” bases allow the creation of an additional stop codon (TAA) at the end of the upstream coding sequence, ending the translation of the desired CDS in accordance with the Marburg Collection design guidelines.

The transcription initiation rate for each RBS element has been specifically adjusted to achieve optimal expression of the desired enzyme within the operon. The relative translation initiation rates for the operon can be observed in the picture below. Where this operon corresponds with the enzymes: CDS_PtaBs , CDS_PKPa , CDS_mdh , CDS_GarK, garR_CDS.

800px-T--MADRID_UCM--RBSPKMCG.png

This polycistronic CDS sequence has been assembled following a modified procedure for golden gate domestication. To know more about the design process of this polycistronic lv.0 parts, visit the iGEM MADRID_UCM 2021 wiki page https://2021.igem.org/Team:MADRID_UCM/Design .

Source

"Gibson assembly using this synthesized basic parts: "BBa_K3726019" "BBa_K3726020""BBa_K3726021"" BBa_K3726022""BBa_K3726023""BBa_K3726024""BBa_K3726025""BBa_K3726026"" ""BBa_K3726027""

References

H. Yu, X. Li, F. Duchoud, D. Chuang and J. Liao, "Augmenting the Calvin–Benson–Bassham cycle by a synthetic malyl-CoA-glycerate carbon fixation pathway", 2021.