Our core wet lab efforts aim to create a minicell-generating BLR(DE3) E. coli strain that produces optically pure L-borneol. To achieve this, we need to first perform gene knockout on the minC, minD, and minE genes of BLR(DE3) to encourage aberrant cell division, leading to minicell creation. After minCDE knockout is performed and a minicell generating BLR(DE3) strain (BLR(DE3)-MC) is established, we performed molecular cloning to assemble the pACYC-LBB and pBAD-LBB plasmids and transformed them into BLR(DE3)-MC to properly establish the MVA and MEP pathways for L-borneol biosynthesis. The resulting BLR(DE3)-MC with pACYC-LBB and pBAD-LBB plasmids has the ability to produce optically pure L-borneol while generating minicells.

Having established a minicell-generating and L-borneol-producing BLR(DE3) strain, we purified L-borneol-containing minicells from the larger E. coli to be used in our water-based insect-repellent fragrance. To test the success of L-borneol biosynthesis and our insect-repellent fragrance’s functionality, we also designed four functional assays: a GC-MS analysis to confirm L-borneol presence, a minicell degradation assay for product longevity, a spray diffusion assay for user comfort and convenience, and a minicell safety assay to ensure biosafety.

This page serves as an overview outlining our experimental plan, to see our experimental results, visit our Engineering page.

To achieve the production of minicells, we first have to perform gene knockout on the production strain of interest: BLR(DE3) (Utilized for its T7 RNA Polymerase system). The knockout of minC (a division inhibitor that prevents the polymerization of the FtsZ protein, ensuring that the division septum forms at the correct mid-cell location.), minD (An ATPase that recruits minC to the membrane and assists in its oscillatory movement between the cell poles.), and minE (Directs the dynamic oscillation of the minCD complex by driving its movement back and forth between the poles, which clears the mid-cell region for correct FtsZ ring formation.) genes of E. coli promotes irregular cell division and minicell formation. The LambdaRED knockout system was utilized for minCDE knockout to produce BLR(DE3)-MC, our minicell-producing E. coli strain.

A diagram of the knockout system is provided below, which shows the steps by which LambdaRED can deactivate certain chromosomal genes (Kenkel, 2016).

For results, visit the Engineering page.

After establishing BLR(DE3)-MC, we performed cloning to assemble the pACYC-LBB and pBAD-LBB plasmids responsible for MVA and MEP pathway establishment. Due to the long length of our constructs, we synthesized them in segments; pACYC-LBB and pBAD-LBB constructs are both divided into 4 separate parts and sent out for synthesis via IDT gBlocks.

To amplify our gBlock construct segments and pACYC & pBAD backbones for assembly later, we first performed PCR using Q5 and PrimeSTAR polymerases.

After successful amplification of pACYC-LBB and pBAD-LBB constructs parts and vectors via PCR, we utilized Gibson Assembly to combine all parts with pACYC and pBAD vectors. The completed plasmids were then transformed into NEB Stable competent cells for the stable propagation of plasmids via cell culturing; successful assembly was confirmed via colony PCR, double digestion, and outsourced sequencing.

To effectively produce our desired enzymes for pathway establishment, we transformed our assembled pACYC-LBB and pBAD-LBB into BLR(DE3) E. coli in order to utilize its T7 RNA polymerase system for better protein production. Since pACYC and pBAD contain IPTG and arabinose inducible promoters, respectively, co-induction was performed by adding IPTG and arabinose concurrently.

After the co-induction of pACYC-LBB and pBAD-LBB in E. coli BLR(DE3), the resulting growth medium will contain an assortment of both minicells and a sizable portion of parental cells. These parental cells are concerns for the biosafety of our product because of our intended application setting: the environment. Therefore, we need a way to extract only the non-replicating minicells to ensure a safe product for human use through purification.

We used a two-step centrifugation method to separate the minicells from the larger parental cells. First, we removed the larger cells and collected the liquid that contained the minicells. Then, the minicells were concentrated, with any remaining debris separated. We filtered the solution using a sterilized filter to further purify our minicells, removing any lingering impurities.

The purified L-borneol-containing minicell can now be incorporated into BOROHMA to create our insect repellent fragrance for safe public use.

Our functional assays are meant to prove that we have indeed biosynthesized L-borneol, but also that the final product is a solution that is convenient and safe for the user.

Even though SDS-PAGE has already been used to determine the existence of all proteins along the L-borneol biosynthesis pathways, L-borneol itself cannot be tested with this technique. Therefore, we enlisted the help of Professor Huang Wei-ning from Yuan Pei University of Medical Technology to verify the existence of L-borneol in our E. coli sample using gas chromatography-mass spectroscopy (GC-MS). We tested the sample against compounds such as chemically synthesized borneol and our solvent for borneol, diethyl ether.

Refer to the Engineering page for the results

To assess whether our fragrance solution will last a reasonable amount of time before its smell-producing active ingredient degrades, we tested how long minicells will stay active by recording the optical density 600 (OD₆₀₀) for around 2-hour intervals and then graphing them to craft a growth curve.

We expect the graph to grow exponentially slowly, meaning that more and more minicells are produced since the E. coli BLR(DE3) divides into minicells, while the number of parental viable cells remains the same.

Refer to the Engineering page for the results

Our mosquito-repelling properties should not detract from the typical properties of perfumes because it would be unfamiliar and unpopular to use from a consumer’s standpoint. Therefore, we used a slow-motion camera to record how the mists of minicells, alcohol, water, and typical water-based fragrances compare.

We aim to prove that there is no difference between the distance and mist spray speed of the minicell-borneol solution with the alcohol, water, and fragrance.

Refer to the Engineering page for the results

The final piece of the functional assay puzzle is solved when we have experimental data to prove the inability of minicells to reproduce in the environment.

To prepare for the assay, we purified minicells by using centrifugation and filtration, which means that there is a very low number of viable parent cells (Barker et al., 1979). Then, we added 200µL of minicell solution at 50 OD₆₀₀ onto a lysogeny broth solid culture and let it incubate for 5 days. A low number of replicating cells will prove that the minicell solution has an extremely low chance of replicating uncontrollably in the general environment.

Refer to the Engineering page for the results

We know there are many more experiments to do and continuations of the project left unexplored. We hope to contribute to future teams by describing the plans listed below.

A common additive in fragrances is a fixative that can aid in the longevity of the smell. Two of these fixatives include Sanatol and Ambergist which are highly sought after and could effectively make our repellent last longer. When GPP is produced from IPP and DMAPP during our borneol production pathway, some of that GPP is converted to FPP by the gene IspA (Wang & Kim, 2015). The converted FPP while useless for creating for creating L-borneol could be useful for Sanatol and Ambergris production through synthetic pathways developed by previous iGEM teams (International Genetically Modified Machine, 2023).

By optimizing their genes for E.coli we can utilize our extra FPP to produce fixatives that will prolong the duration of both our repellent and fragrance (Zhang et al., 2022).

Geraniol is a natural compound found in various essential oils, particularly rose and geranium. It is known for its pleasant, floral scent and is commonly used in perfumes, cosmetics, and food flavoring. This compliments borneol which has a stronger woody scent. Additionally, using geraniol alongside borneol may have a synergistic effect in terms of both repellency and fragrance longevity. Studies conducted on naturally obtained repellent chemicals have revealed that when several terpenes are mixed, they tend to last longer than usual because mosquitoes are said to adapt to one scent at a time (International Genetically Modified Machine, 2023). This combination of geraniol and borneol could lessen the risk of using synthetic chemicals thereby delivering the product in an environmentally friendly manner.

By introducing the gene encoding geraniol synthase from Ocimum basilicum, which is responsible for converting GPP to geraniol we can produce both L-borneol and our fragrance at the same time solidifying our product as a repellent fragrance. The enzyme geraniol synthase works by removing the diphosphate group from GPP, generating geraniol (Liu et al., 2016).

Our fragrances' would benefit from EPS because they are useful biomaterials for individual cells to cling onto organic cloth, which would increase the longevity of the mosquito repellent and fragrance smell. These saccharides are coded by the yjbEFGH genes, which we plan to overexpress in the E. coli BLR(DE3) cell (Ferrières et al., 2007).

We plan to transfer the plasmids of the E. coli BLR(DE3) strain into ClearColi™ for an even more robust biosafety system. ClearColi™ is a genetically engineered E. coli BLR(DE3) where its surface toxins have been completely removed, creating a cell membrane without any toxic ligands or other toxic extracellular molecules (Mamat et al., 2013).

It further reinforces the biosafety mechanism by eliminating all membrane toxins, allowing for high-concentration use of the fragrance.

While SDS-PAGE provided a preliminary reading for our protein quantification, the next step for this task would be to perform a Bradford assay, which uses a spectroscopic analytical procedure to measure the concentration of protein in a solution.

Performing a Bradford assay would make our production release model a lot more robust with precise and accurate data to model.

Proteomic assays on the validation of proteins would allow us to determine with certainty the existence of the proteins along the MVA, MEP, GPP, and L-borneol synthesis pathways. Looking for unexpressed proteins, we know which sequences to troubleshoot or replace and redo the experiment with.

Finally, with the corrected proteins, we could also use ethyl acetate for L-borneol extraction from minicells for GC-MS assay, which is a more fitting organic solvent for L-borneol due to its logP value.

Our GC-MS experiment led us to conclude that BbTPS3 may have been misfolded or mismodified which resulted in a buildup of GPP-like substances and a lack of any molecules downstream (Ma et al., 2022). By diagnosing each protein individually by checking its substrate-to-product conversion, we can see which proteins were misfolded and modified to see which genes to treat or replace.

Due to the high demands that we place on our E. coli, we hypothesize that metabolic stress might hinder the effective biosynthesis of L-borneol. To diagnose the metabolic stress possibility, we will use metabolic flux analysis, which would help trace GPP's fate and minimize the formation of byproducts. This would optimize L-borneol production, hopefully creating a better environment for biosynthesis.