Detailed, step-by-step methods for constructing, transforming, and extracting terpenoid alcohols using Saccharomyces cerevisiae.
Purifying an alcohol of interest from any host organism first requires designing or identifying a genetic construct that codes for the desired enzymes involved in the biosynthesis of these alcohols and contains the conditions for these proteins to be expressed. For our project, we aimed to express two different enzymes so we considered designing and assembling two distinct genetic constructs, but eventually identified an existing construct that contained both of our desired enzymes. In a selected construct you should ensure compatibility with your selected vector and growth strain and expression under your desired promoter(s). After the selection of your genetic construct. Once you have identified your desired construct you need to create competent cells to express your construct. From your competent cells you can then isolate the DNA and move on to transform your host organism!
Following the successful identification and isolation of the genetic construct, the project proceeds to the transformation of the host organisms. This stage is pivotal in the context of our investigation, especially since our end goal involves the production of alcohols, thus our host organism should contain at least part of the desired biosynthesis pathway.
First the genetic construct is verified so that the project will move on. In our project we decided to verify the isolation using a southern blot and so the protocols are tailored accordingly. After confirming successful isolation of the desired construct, the host transformation protocol is initiated. Leucine-deficient Saccharomyces cerevisiae is cultivated and transformed. Here the host selection and construct identification play a very important role since for the host organism to be able to take up the construct, the plasmid should express an essential amino acid which has been deleted from the host organism. Furthermore, when inserting a construct that contains only part of a biosynthesis pathway, like in our project, you need to ensure that your host organism contains the remaining pathway. This forms the basis for the next steps of the experiment which involves the extraction of the desired alcohols.
For the final step of our investigation is the extraction of the terpene alcohols from the host organism. We wanted our solution to be scalable, so we landed on a method that prioritized the preservation of the yeast for the continued production of our desired alcohols. We decided to add olive oil as an organic solvent halfway through the fermentation process to trap the alcohol products.
If you choose to follow this method, after 4 days of fermentation you should extract the organic solvent to obtain the two alcohols. After extraction you can purify the product. We decided to follow a funnel separation protocol for our purification. Further for confirmation you can measure the absorbance of your sample under UV: 200 nm for linalool confirmation and 280 nm for nerolidol confirmation.
As high school students diving into synthetic biology for the first time, we strove to understand fundamental concepts in order to design an appropriate method for our investigation. In this effort, we created a guide to help other first-timers grasp key concepts they can build upon for their own projects.
This guide walks through core ideas like genetic constructs, chassis selection, transformation, metabolic pathways, and verification methods — bridging the gap between classroom theory and real wet lab practice.
The dry lab focused on simulating enzyme–alcohol interactions through structure prediction, molecular modeling, and docking simulations. Enzyme structures were predicted from amino acid sequences obtained from protein databases using SWISS-MODEL, and models were evaluated using GMQE and QMEAN scores to ensure structural correctness.
Ethanol, linalool, and nerolidol molecules were built and optimized in Avogadro using three-dimensional modeling and force field minimization (MMFF94) to geometrically optimize and pre-prepare ligand structures for docking. The target for GABA–alcohol docking simulations was either the GABA receptor or the active site of interest to the enzyme, and optimized alcohol molecules were defined as ligands.
Autodocking was performed using AutoDock Vina with a grid box placed over the active site of the receptor, predicting multiple binding poses and ranking them based on binding affinity (kcal/mol). Top-ranked interaction analyses of complexes were performed using visualization tools such as PyMOL and Chimera, and further validated using PLIP for detecting hydrogen bonds, hydrophobic contacts, and π–π interactions stabilizing binding.
Finally, in order to ensure reproducibility, all input and output files, parameters, and software versions (SWISS-MODEL, Avogadro, AutoDock Vina) were recorded methodically so that enzyme–alcohol docking and simulation data could be reproduced consistently.
This protocol details the computational workflow used to model and analyze enzyme–alcohol interactions, focusing on GABA–alcohol complexes.
To mimic enzyme–alcohol interactions by predicting structures, performing molecular modeling, and running docking simulations, with a focus on GABA–alcohol complexes.