Part:BBa_K1493300

methionine-γ-lyase

Is an enzyme that converts L-methionine to methanethiol, ammonium and 2-oxobutanoate. This biobrick and synthesized and codon optimized for Pseudomonas putida based on the sequence of Brevibacteirum linens who's enzyme has shown to have high affinity towards the side of methanethiol. When methanethiol is formd it gets further oxidezed to dimethyldisulfide(DMDS) and dimethyltrisufide(DMTS). More information can be found on our wiki.

Characterization

The biobrick was tested in a shuttle plasmid (SEVA) and was coupled behind an IPTG inducible promoter. Plasmid was transformed in P.putida KT2440. Enzymes were then isolated via sonification, enzymes were then used in an assay. MDS and DMTS were suppose to be measured via Gas Chromatography (GC).But it was not possible due to unexpected problems with the GC machines and are planed to be done after wiki-freeze. However an in vivo assay was done anyway, co-inoculating our transformed P.putida with Fusarium TR4.

Fig 9.In vivo assay, P.putida (Methionine-γ-lyase) co-inoculated with Fusarium. Red circle indicates area occupated by Fusariumgrowth. Foc control=Fusarium oxysporum cubense TR4, WT=wild type P.putida KT2440 and MgL= P.putida with an overexpression of Methionine-γ-lyase.

With the in vivo assay it can be seen that the tranformants containing Methionine-γ-lyase has better inhibition when compared to the wild type P.putida. leading for us to believe that there is an increase of DMDS and DMTS production.

Improvement for the purification

Group : iGEM18_Aix-Marseille

We added in c-ter a histidine tag (BBa-K2718005).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 154
  Illegal NgoMIV site found at 667
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 460