We designed 93 parts to accomplish it this year. Among them, 40 parts are composite parts and the rest are basic parts. We gave experimental characterization of some important parts. All parts that we used had been submitted to the iGEM Registry following the BioBricks assembly standard. This part collection contains plasmids we designed by the exogenous enzyme genes during our experiments. Each part could accomplish one step from the Δ-9 elongase/desaturase pathway by integrating the linear DNA fragments into the the genome of the Engineered Y. lipolytica, as well as indicate success of integration by the URA/Leu gene since the Yeasts we used were nutrition-deficient (uracil or leucine deficient) strains. The part collection includes the most of transcription units we have validated which expresses the high enzymatic activity, from our pathway choosing to the optimizing phase of increasing production. Here is a table containing a brief description of our parts. Our favorite basic parts is BBa_K4343113, and the favorite composite parts are BBa_K4343071. If you want to get more detailed information about our parts, we strongly recommend you to visit the corresponding parts pages on the iGEM BioBricks Registry.

Basic Parts

Among the 53 basic parts we designed this year, BBa_K4343113 (Sc4Des) is our favorite part. The length of Sc4Des gene is 1284bp (Fig. 1). The Sc4Des gene encodes Δ-4 desaturase from Schizochytrium sp., which could convert C22:5 to C22:6 in the last step of Δ-9 elongase/desaturase pathway to synthesize DHA.

Fig.1 The Gel Electrophoretic Image of Sc4Des gene

Composite Parts

We designed a series of composite parts and tested most of them to insure the reliability. We like BBa_K4343071 best since it was designed based on our favorite basic Part.

We constructed a plasmid containing promoter TEF, Sc4Des, and terminator Cyc1t through the T4 ligase. The plasmid was linearized by enzyme cleavage and then transformed into engineered strain with one copy of TsElo5.

The Sc4Des gene in the plasmid encodes Δ-4 desaturase from Schizochytrium sp., which could convert C22:5 to C22:6. After integrating the linear DNA fragments into the genome of the engineered Y. lipolytica strain Po1f-20, the percentage of DPA and DHA in the engineered strain Po1f-21 produced were 2.7% and 18.9% of TFAs respectively， which the percentage of EPA was 2.5% (Fig. 3).

Fig.2 Construction of plasmids of pUC-HUH-Sc4Des

Fig.3 (A) Fatty acid profiles of the Po1f-20 and Po1f-21 determined by Gas Chromatography analysis. (B) Different fatty acid percentage of TFAs in Po1f-20 and Po1f-21.