Coding

Part:BBa_K3866023

Designed by: Pericles Vasileiou   Group: iGEM21_Thessaly   (2021-09-24)
Revision as of 21:05, 25 September 2021 by Periclesv (Talk | contribs) (→‎Verification of cloning)


sbm - Methylmalonyl-CoA mutase GB compatible with B2-B5


Figure 1. The level 0 module : pupd2- sbm (illustration from SnapGene)

Usage and Biology

Catalyzes the interconversion of succinyl-CoA and methylmalonyl-CoA. Could be part of a pathway that converts succinate to propionate.

Design Notes

The sequence was domesticated. We removed BsmBI, BsaI, BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in pUPD2 BBa_K3505007 and has overhangs compatible for Golden Braid cloning. This has position B2-B5.

Figure 1.The overhangs of this part in the GoldenBraid Grammar

Verification of cloning

Figure 3. (C= Cut, U=Uncut) Restriction digestion of pTrc:RBS (C1 + C2) with: EcoRI + EcoRV, Expected bands: 1280bp, 896bp, Positive result: C1 + C2

Source

Synthesized by IDT.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1697
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1697
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1697
    Illegal BamHI site found at 985
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1697
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1697
    Illegal NgoMIV site found at 2012
    Illegal AgeI site found at 149
  • 1000
    COMPATIBLE WITH RFC[1000]

References

Haller T, Buckel T, Rétey J, Gerlt JA (2000). Discovering newenzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli. Biochemistry, 39:4622–4629. https://doi.org/10.1021/bi992888d

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