Figure 1: Original plan for composite parts

In conjunction with the composite part, we had planned to screen various compounds that where kindly given to us to by Givaudan as reference points, as the compounds we were producing with our biobricks matched the reference compounds we had been given, giving us the opportunity to compare our results to them.  On top of this it was hoped that an increase in the concentration of substrate could be used by the enzyme in the E.coli by introducing the Mevalonate pathway from plants, which in the scale up we would hope would increase our yield.

In summary during this project, we have designed new biobricks containing [the enzymes] that was subsequently synthesised. We then successfully constructed a new basic biobrick part containing R – Linalool synthase using the Gibson assembly method. We have caracterised this part using Gel electrophoresis.

We firstly optimized sequences containing R-linalool and Zingiberene and these genes were synthesized by Life technologies. We opted to use Gibson assembly method in the assembly of the linearized sequence fragments onto the standard RFC10 compatible backbone pSB1C3. We were then able to use Gibson assembly to successfully place R-linalool into pSB1C3 as well as Limonene from pSB1C3 onto pSB1A3 (Figs 1 and 2)

Figure 1. Agarose gel electrophoresis of double digest of plasmid containing the Limonene Synthase gene (BBa_I742111) using enzymes EcoR1 and PsT1. From left to right, the lanes show DNA ladder, LS double digest, control. This experiment show successful assembly of Limonene synthase into pSB1A3 

Figure 2. Agarose gel electrophoresis of double digest of plasmid containing the R-linalool Synthase gene (BBa_K1454000). From left to right, the lanes show DNA ladder, RL double digest, control.The digestion was done using EcoR1 and PsT1. This experiment shows the successful Gibson assembly of our newly designed biobrick gene for R-linalool onto pSB1C3.

Upon the successful assembly of our new R-linalool biobrick, a mini-prepped plasmid sample was seqenced. As can be seen in Figure 3, using Clustal Omega we have confirmed that our biobrick contains the optimized gene sequence of R-linalool synthase.

Our Part:

R-Linalool Synthase: BBa_K1454000

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