Part:BBa_K1736200
Harmonised Ethene Monooxygenase
This construct was the main component of the project which would be responsible for the expression of ethene MO enzyme and the biosynthesis of ethene oxide from ethene. After performing successful Golden Gate cloning (putting 3 Gblocks into a vector, you can find more information about our method here), we transformed it into P. putida competent cells and observed no ethene oxide after incubating the sample in the presence of ethene through utilising the qualitative NBP assay. We believed that the constitutive expression of ethene MO straight after transformation would put too much stress on the cell, hence, we introduced LacI to control the expression of the system. However, after IPTG induction, the NBP assay failed to detect any traces of ethene oxide, which shows that the inactivity lies within the enzyme itself, not the cells. One reason for the ethene MO inactivity could be that our inhouse harmonisation tool TransOpt is not able to yield active and properly folded proteins (through changing the codon sequences) as confirmed in the experimental validation with BsFP (for more information on the experimental validation, visit results and discussion). This conclusion that the non-functionality of the genetically modified P. putida host due to the ethene MO inactivity is further supported by the fact that the previous ethene MO constructs came positive and the NBP assay was reliable when tested with epoxides. While this particular construct and sequence of ethene MO was not functional, future iGEM teams should make use of the method used and the harmonisation considerations taken into account to make effort for the development of a functional ethene MO. Important notes concerning this part: pSB1C3-EtnABCD part submitted is missing the XbaI cut site at the BioBirck prefix. A mutation (D91E) exists in the sequence due to accidental change while removed BsaI cut sites, however, the change is conservative and is present in ethene MO of other Mycobacterium strains, which means that it is unlikely to have had an effect on the function of the enzyme.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 554
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 119
Illegal BglII site found at 3698
Illegal BamHI site found at 254
Illegal BamHI site found at 969
Illegal BamHI site found at 1887 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 146
Illegal NgoMIV site found at 1717
Illegal NgoMIV site found at 2286
Illegal NgoMIV site found at 2825
Illegal NgoMIV site found at 3246
Illegal AgeI site found at 3949 - 1000COMPATIBLE WITH RFC[1000]
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