Once we had our inserts designed, we had to modify our target vector to accept them. The inserts we had designed for the menthol pathway had been engineered with novel ribosome binding sites, so we amplified around our expression vector's ribosome binding sites. This contrasted with the insert for our linalool pathway, which was designed to rely on the ribosome binding site in the vector itself.

Figure 1: Gel electrophoresis of the menthol and linalool vectors

Lanes 2 and 3 contain our menthol vector, while lanes 4 and 5 contain our linalool vector. We expected them to locate to the 5.1 kilobase (Kb) pairs mark, so from this gel we moved ahead with the first clone of the menthol plasmid and both clones of the linalool vector.

Now that we had modified PET28a to be ready to accept our inserts, we were ready to continue with our cloning process and begin moving our inserts into PET28a. We performed Gibson assembly with both our menthol and our linalool constructs and then transformed them into competent DH5a cells. After the transformation we selected 6 colonies from each Gibson assembly plate and performed a colony PCR. Our menthol inserts totaled to be 5.4 kB and our linalool insert totaled to be 1.8 kB.

Figure 2: Gel electrophoresis image to confirm our colony PCR for both menthol and linalool constructs

The menthol colonies are on the left, the ladder is in the middle, and the linalool colonies are on the right. From this result we can see that linalool clones 1,2 and 4 were successful, while none of the Menthol constructs were successful.

Faced with an unsuccessful Menthol assembly we attempted the synthesis once more and this time we picked 30 colonies. Again, the menthol constructs are expected to migrate to 5kb.

Figure 3: Image of two gels that were used to check if our re-run of menthol Gibson assembly was successful

The colony in lane 6 in the first gel and the colony in lane 9 on the second gel were successful. While this was a successful assembly, we unfortunately lost the colony corresponding to that DNA, so we were unable to proceed with assembly.

For this project we set out to engineer E. coli bacteria to produce menthol and linalool from the precursor plasmid pJBEI-6409. While we were able to engineer our E. coli with the necessary enzymes to produce Linalool, we did not observe any Linalool production. On the other hand, while we did have success engineering the menthol pathway, we suffered a protocol breakdown that led to us losing the colony that contained the pathway. Thus, we would like to lay out some protocol and experimental changes in order to improve production.

The unsuccessful creation of the menthol pathway was a blow to the team. This was especially difficult because we were able to visualize the pathway several times on a gel, we were just never able to collect the colony afterwards. Moving forward we will modify our colony PCR protocol to include dissolving the colony in water in a tube labeled with colony number and date. And then storing all the tubes from that day on one plate, labeled with the date. Thus, after the colony PCR gel is run, we would be able to return to the rack and start overnight cultures from the colony number which corresponds to the colony on the gel.

For starters we would recommend cutting of the first 67 amino acids from the Linalool Synthase, stopping at the double arginine residues at position 68 and 69. Previous research has shown that the first 67 residues are not important to produce linalool and instead are targeting sequences, intended to target the protein to the plastid organelle in plants (Bohlman et al. 1988). Removal of this sequence has resulted in higher yields when the linalool synthase has been expressed in Yeast (Zhang et al. 2022).

The precursors were produced by pJBEI-6409, a plasmid containing an engineered MEV pathway for the production of Limonene in E. coli. This plasmid is under Lac/IPTG control with very specific induction conditions. We produced our enzymes from a pET28a(+) vector under Lac/IPTG control. This means that we did not have individualized control over the plasmids. For future experiments we recommend transitioning our enzymes to an arabinose inducible promoter, to provide independent control over the plasmids in the cell.

Our biggest question mark while performing these experiments was whether we would locate our compounds in the supernatant or in the cell lysate. The literature was unclear as to this answer. For a further step we would thus suggest analyzing the cell lysates as well as the cell supernatants, to fully visualize the amount of compound being produced by the cell. Work by previous iGEM teams supports this hypothesis, iGEM_2020 Keystone produced linalool in E. coli and were able to visualize by extracting with hexane.

Finally, for our Menthol pathway we performed ribosome binding site sequence optimization to achieve the highest levels of RBS translation. For some proteins there had been previous work which had suggested optimal molar ratios (Yoshida et al. 2021, Currin et al 2018). But for others we had to guess translation rates. For the next step we would recommend for each protein designing three RBS sequences, one “low”, one “medium” and one with “high” expression and testing each for production. This same proccess could also be applied to the linalool pathway with a similar goal.

Zhang, Y., Cao, X., Wang, J., & Tang, F. (2022). Enhancement of linalool production in Saccharomyces cerevisiae by utilizing isopentenol utilization pathway. Microbial cell factories, 21(1), 212.

Bohlmann, J., Meyer-Gauen, G., & Croteau, R. (1998). Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proceedings of the National Academy of Sciences of the United States of America, 95(8), 4126–4133.

Yoshida, E., Kojima, M., Suzuki, M., Matsuda, F., Shimbo, K., Onuki, A., Nishio, Y., Usuda, Y., Kondo, A., & Ishii, J. (2021). Increased carvone production in Escherichia coli by balancing limonene conversion enzyme expression via targeted quantification concatamer proteome analysis. Scientific Reports, 11(1), 22126.

Currin, A., Dunstan, M. S., Johannissen, L. O., Hollywood, K. A., Vinaixa, M., Jervis, A. J., Swainston, N., Rattray, N. J. W., Gardiner, J. M., Kell, D. B., Takano, E., Toogood, H. S., & Scrutton, N. S. (2018). Engineering the “Missing Link” in Biosynthetic (−)-Menthol Production: Bacterial Isopulegone Isomerase. ACS Catalysis, 8(3), 2012–2020.