We successfully assembled our constructs and transformed E. coli BLR(DE3) with pACYC-Duet (pACYC) and pBAD-24 (pBAD) plasmids that contain L-Borneol Biosynthesis (LBB) genes to form pACYC-LBB and pBAD-LBB. The success of plasmid combination and protein expression was confirmed using colony PCR, double digestion, and SDS-PAGE analysis. Finally, we were able to detect the presence of many potential geranyl diphosphate-like compounds through GC-MS; with our future plans, we are confident that we can produce L-borneol with the modification of a few enzymes in the biosynthesis pathways.

The pACYC-LBB and pBAD-LBB plasmids were designed using Benchling, and IDT synthesized the DNA sequences of the plasmid. Due to the length of the construct, it was synthesized in four fragments, with breakpoints at 3065 bp, 6065 bp, 9065 bp, and 11744 bp. We utilized a dual promoter strategy to optimize protein expression for L-borneol production.

Proteins that require overexpression, such as Dxs, Dxr, HmgR, IDI, IspA, LBPPS, ALP, and Hmg, are placed into the pACYC backbone to exploit its efficient T7 promoter. While this promoter is known for its transcription strength, it is not ideal for expressing long sequences exceeding 3 kb. We addressed this limitation by introducing additional promoters for every 3 kb or more within the construct, ensuring effective transcription of each segment. The araBAD promoter was used for proteins that require lower or moderate expression. The pACYC-LBB plasmid carries resistance to chloramphenicol, while the araBAD plasmid carries resistance to kanamycin. This differentiation between antibiotic resistance markers facilitates cloning, selection, and easy identification of transformants containing either plasmid.

In the construction process, we extracted the usable pACYC backbone from the original pACYC plasmid by performing double digestion using the restriction enzyme DpnI, cleaving the undesired methylated backbone while retaining the desired unmethylated pACYC backbone. Each protein is expressed independently to ensure flexibility and proper folding, avoiding challenges associated with fusion proteins and allowing individual fine-tuning.

We hypothesize that employing a dual plasmid system with distinct promoter controls can optimize the production of L-borneol in E. coli BLR(DE3). Balancing the inducible gene expression with different promoter strengths will mitigate potential metabolic stress on E. coli while providing flexibility in optimizing production levels.

We designed our experiments into four parts: minicell building through minCDE knockout, pACYC-LBB building and transformation, pBAD-LBB building and transformation, and effectiveness testing.

We engineered E. coli BLR(DE3) to produce minicells by knocking out the minCDE genes using the Lambda Red recombineering system. The process began by introducing the pKD46 plasmid into the strain, which enabled the expression of the Lambda Red recombination proteins (Exo, Beta, and Gam) under arabinose induction. These proteins were essential for facilitating homologous recombination. To target the minCDE genes, a double-stranded DNA (dsDNA) substrate was designed with an ampicillin resistance cassette from the pKD13 plasmid, flanked by homology arms that matched the sequences adjacent to the minCDE operon in the genome. This dsDNA was then introduced into the E. coli BLR(DE3) cells through electroporation, allowing the Lambda Red proteins to mediate the recombination event, replacing the minCDE operon with the ampicillin resistance gene.

Following electroporation, the cells were selected on ampicillin-containing plates, and colony PCR was performed to confirm that the minCDE operon had been successfully replaced by the resistance marker. To remove the antibiotic resistance gene, the pCP20 plasmid was introduced to express FLP recombinase, which excised the ampicillin cassette, leaving behind a clean knockout of the minCDE genes. Finally, the success of the knockout was confirmed by sequencing the E. coli BLR(DE3) genome to verify the deletion of the minCDE operon. Microscopically, we observed small, spherical minicells—which lack chromosomes—alongside larger, rod-shaped parental cells, confirming the effective disruption of cell division as shown in the microscope image below.

This knockout system ensures that the engineered strain produces minicells, which are valuable for a variety of applications, such as drug delivery or studies of cell division. By knocking out the minCDE genes, we successfully created a strain that exhibits clear morphological distinctions between minicells and parental cells, providing a strong foundation for the subsequent use of minicells in encapsulating borneol, offering great potential for efficient and safe delivery of L-borneol.

Amplification & Gibson Assembly

Four plasmid inserts (pACYC-LBB's Parts 1~4) were acquired via IDT gBlocks synthesis. For cloning purposes, we planned to assemble the plasmids separately in different E. coli cultures for easier troubleshooting and cloning.

Parts were made in 2.7~3k segments according to IDT's guidelines on gBlock submissions. When we received the gBlocks, we performed polymerase chain reactions (PCRs), which provided ample parts to work with.

Calculations were made with the concentration data from the microprep stage to approximate the 3:1 insert-to-vector ratio by mass. We built the plasmid using the Gibson protocol, and then the ligation product was transformed into NEB Stable and later DH5α for cloning.

ColonyPCR

To verify the accuracy of the assembly, we employed two methods: double digestion and colony PCR. However, the initial results of colonyPCR show inconsistencies between the expected and observed fragment sizes. We hypothesized that the false positives were due to improper handling of the template DNA during gel extraction of the PCR-amplified backbone, which may have allowed the uncut template to contaminate the Gibson assembly and transform into E. coli BLR(DE3), giving it antibiotic resistance without the edited multiple cutting sites (MCS).

After addressing these issues by cleaving all unedited plasmid backbones, we successfully performed Gibson assembly, resulting in a construct of the correct size. We then transformed the assembled plasmid into E. coli BLR(DE3) via heat shock and redid the colony PCR protocol to produce a successful result.

Double Digestion

To further confirm that we assembled all four parts of the fragments properly, we performed double digestion to make sure that the plasmids were assembled properly. We double-digested and split the 15k plasmids. Using Gel electrophoresis we confirmed that the length of the fragments is the expected 6k and 9k bands. We used numerous restriction sites over many trials and consistently got a successful result.

Amplification & Gibson Assembly

Cloning of pBAD-LBB fragments was made in a similar process to that of pACYC-LBB, except that we switched in favor of the higher quality PrimeSTAR polymerase to amplify the long fragments to save time and resources. Parts were spit on the bases of 3k bp and the remainder, leaving three parts of 3kbp and one part of 2.7kbp

After ensuring that the PCR fragments were the right lengths, this plasmid underwent the same processes as pACYC-LBB of Gibson assembly and chemical transformation.

ColonyPCR

ColonyPCR was performed on picked colonies from a solid culture plate. We chose two primers, one forward and one reverse at the start of part 2 and the end of part 3. Even when we chose primers to conduct colony PCR at 2000 bp, the result still deviated from our expected result.

After multiple attempts at colony PCR, we were unable to determine the correct sequence. This prompted us to do some analysis on the issue, finally diagnosing and learning about the reasons for the failure. Therefore, we moved on to other methods of plasmid validation, such as double digestion.

Double Digestion

We performed double digestion with a variety of restriction enzymes that split the 15k plasmids into 6k and 9k for all trials. After gel electrophoresis of the cut plasmids, we ensured that the plasmid was successfully made to the right length. Similar to pACYC-LBB, double digestion of the pBAD-LBB plasmid consistently yielded successful results.

After the successful confirmation of plasmids separately in competent cells, the individual-plasmid-containing E. coli DH5α competent cells were lysed, and the collected plasmids were transformed again, this time with two plasmids in one cell.

MVA, MEP, GPP synthesis, and L-borneol synthesis pathways proteins were expressed in 400 ml of LB broth with E. coli BLR(DE3) containing both plasmids. After literature research, it seemed apparent that the expression of the plasmid’s genes was optimal in a specific condition; we’d follow Zhangbao Wang et al.’s protocol at all points if possible. Plasmid induction was performed at two temperatures, 37°C until the OD₆₀₀ value hit 0.6, then transferred to 25°C for overnight induction with IPTG and arabinose.

The OD₆₀₀ test for E. coli BLR(DE3) provided two results. One is that the induction of proteins in the plasmids was successful, as determined from the lower growth curve shown for induced cells. It also tells us that we performed a successful minCDE knockout procedure given the exponential growth curve.

While polypeptides were confirmed to be present through protein expression assays, their function might not be active, which Professor Wei-Ning Huang led us to realize, due to incorrect polypeptide modification post-translation and incorrect protein folding due to foreign mediums of modification. Therefore, we took several measures to validate protein expression.

SDS-PAGE

After our induction of cells with IPTG and arabinose, 20µL of 0.3 OD₆₀₀ minicell solution was used to perform SDS-PAGE to determine the existence of the proteins that make up the pathways of L-borneol production. For both pACYC-LBB and pBAD-LBB, the proteins were present, which confirmed the theoretical biosynthesis of L-borneol. We anticipated that the following proteins would be present in the associated Dalton number, in the order of placement in our plasmids: pACYC-LBB with 1-deoxyxylulose-5-phosphate synthase (Dxs), 68 kDa; 1-deoxyxylulose-5-phosphate reductoisomerase (DXR), 43 kDa; isopentenyl diphosphate isomerase (IDI), 38kDa; farnysyl diphosphate (IspA), 32 kDa; L-bornyl diphosphate synthase (BbTPS3), 64.5 kDa; alkaline phosphatase (ALP), 49.5 kDa; 3-hydroxy-3-methylglutaryl-CoA reductase (HmgR), 115 kDa; pBAD-LBB with acetoacetyl-CoA thiolase (ACCT), 41.5 kDa; 3-hydroxy-3-methylglutaryl-CoA synthase, 55 kDa; mevalonate kinase (MK), 48.5 kDa; mevalonate-5-phosphate kinase (PMK), 50.5 kDa; mevalonate-5-diphosphate carboxylase (MDD), 44 kDa; 4-diphosphocytidyl-2C-methyl-d-erythritol cytidylyltransferase (IspD), 26 kDa; 4-diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE), 31 kDa; 2C-methyl-d-erythritol-2,4-cyclo-diphosphate synthase (IspF), 17 kDa; 4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate synthase (IspG), 41 kDa; 4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate reductase (IspH), 35 kDa.

Re-attempt

Our first attempt at SDS-PAGE was unsuccessful due to a wrongly chosen gel that did not fit our required parameters. At 8% gel concentration, which had its smallest band at 35 kDa, the gel did not provide enough specificity for our needs to validate the existence of several enzymes under the 35 kDa mark. Learning from this test, we then chose another concentration of gel (at 10% and 12%), which gave us more bandwidth to work with for the validation of the production of proteins from the 8-9kDa to 35kDa range.

After verifying the production of proteins coded by the plasmid, we also analyzed a pure and quantitative marker called bovine serum albumin (BSA) for the quantification of proteins using computer software called ImageJ.

Visit the Production-Release Model in our Model page to learn more about the ImageJ quantification of our proteins.

Having a cell culture containing both of our desired plasmids and having checked protein expression and plasmid accuracy, we then sought to confirm the production of L-borneol through another means. GC-MS was utilized to determine the molecular composition of certain compounds (including L-borneol and GPP-like fatty acid compounds) in our induced sample.

We discovered that there are a plethora of compounds similar to geranyl diphosphate (GPP). GPP is not expected to appear due to its high polarity, preventing it from being loaded into the detection instrument that only allows non-polar vapors to pass into it, so similar compounds to it point towards the successful production of GPP. The presence of GPP-related byproducts indicates that the pathway is partially functional, providing promising signs for further optimization.

The following list describes how some products may have been formed:

• Und- (RT 6.2) Dode- (RT 13.5) Penta- (RT 22.2) decanes: Appears to be fatty-acid-like; the chemical structure of these -decanes hint towards a GPP precursor or derivative, again through non-specific enzymic or environmental means.
• Cis-3-Tetradecene (RT 13.2): Potentially a derivative of isoprenoid metabolism. This compound is determined to be the most similar GPP precursor/derivative. It was likely produced through non-specific processes.
• Butylated Hydroxytoluene (BHT, RT 27.0): This compound originates from the diethyl ether solvent background and is not a target product. This was concluded because of an MS test on diethyl ether.

Many fatty acids may have formed because GPP is a reactive intermediate that may undergo isomerization or conversion by other enzymes, leading to the formation of byproducts such as cis-3-tetradecane, undecane, and other alkanes and alkenes. Due to its high polarity, GPP is not easily detected by GC-MS. Since GPP was not converted into L-borneol at a detectable concentration, its double-bonded structure may have undergone diversion into general metabolic pathways, resulting in the formation of linear and branched alkanes and alkenes that resemble GPP.

Learning from the results, we thought that downstream enzymes like BbTPS3 failed to convert GPP efficiently enough. Additionally, the non-specific compounds detected may have arisen from fatty acid metabolism pathways.

Purification of BOROHMA Minicell

Minicell purification is a crucial part of our project, ensuring biosafety and yielding clean samples for functional assays. After the induction period, we employed a two-step centrifugation process to isolate the minicells from the parental cells.

In the first step, the solution was spun at 2000xg for 20 minutes, which pelleted the larger parental cells. The supernatant, containing the minicells, was carefully collected. In the second step, the supernatant was centrifuged at 13000xg for 30 minutes, effectively pelleting the minicells and separating them from any remaining cellular debris.

To further purify the minicells, we applied a filtration step. The minicell solution was passed through a sterilized filter to remove any residual contaminants and ensure biosecurity. A syringe was used to push the growth media through the filter attachment, further refining the purity of the minicell solution. Through this combined centrifugation and filtration process, we achieved a highly purified sample of minicells, effectively separated from parental cells and ready for use in downstream functional assays.

Through these two purification mechanisms, minicells are separated from the parent cell with extremely high purity.

With these preparations made, we performed 2 more sets of functional assays in addition to precise evidence from GC-MS that gave us parameters to create our product.

Spray Diffusion Assay

We tested the distance and speed of the mist coming from controls and variables such as 2mL 95% alcohol, 1mL 95% alcohol combined with 1mL ddH2O, 2mL ddH2O, 2mL saturated 70% L-borneol in 95% alcohol, and 2mL of minicell solution at 50 OD₆₀₀. Through our spray testing experiment, we determined that the distance and speed at which the solutions traveled showed no significant difference, leading us to conclude that there is a difference between the way that consumers would use our minicell product versus other conventional perfume types.

Minicell Biosafety

The minicell biosafety assays are quintessential to ensuring the safety of our environment as well as our skin. There are several ways to test for the successful knockout of the minCDE genes. As seen above, one way is to see the cells through a light microscope, which gives physical proof that there are minicells present in the solution. We also used another method of assay where we placed 200µL of ultra-pure minicell solution into an agar plate. After five days, we observed one colony growing on the plate, which means that the biosafety mechanism was executed successfully.

As mentioned earlier, when performing heat shock transformations, we encountered many false positive results where blank plasmids were transformed into competent cells, which gave it antibiotic resistance even though its MCS was not assembled with the correct parts.

From this, we learned that we needed a way to prevent the misassembled plasmids from being transformed into cells. These misassembled plasmids, which have the potency to be expressed in a cell when transformed, came from the original template DNA, which has methylated groups due to the inherent nature of organic plasmid materials.

To address this issue, we employed DpnI, an enzyme that selectively digests methylated and hemimethylated DNA. By utilizing DpnI, we effectively eliminated the remaining template DNA, ensuring that the template DNA utilized in our Gibson assembly was free from contaminants.

Enzyme Mismodification and Misfolding

Based on the successful SDS-PAGE results and the unsuccessful GC-MS results, we infer that errors may have occurred in the secondary structure of proteins. Although the efficient production of borneol relies on all parts of the MVA, MEP, GPP, and L-borneol synthesis pathways, the upstream MVA and MEP pathways are present in E. coli, albeit at low levels. The bottleneck appears to be at the BbTPS3 enzyme, which converts geranyl diphosphate (GPP) into L-bornyl diphosphate.

We hypothesize that the lack of BbTPS3 activity stems from two major issues: insufficient post-translational modifications and protein misfolding. Since BbTPS3 is a eukaryotic gene from Blumea balsamifera, E.coli may lack the necessary mechanisms to carry out the post-translational modifications required for enzyme activation. Furthermore, the prokaryotic environment of E. coli may not provide the proper conditions or cofactors for correct protein folding, rendering BbTPS3 inactive.

To ameliorate this concern, protein expression analysis using Western blot will help verify proper enzyme production. Additionally, we will perform control experiments using non-induced E. coli BLR(DE3) and strains without the target gene to identify naturally occurring compounds versus non-specific byproducts from pathway induction.

Enzyme Mismodification and Misfolding

Another possible explanation for the failure to produce L-borneol is metabolic stress. Induction of foreign gene expression could have placed undue stress on E. coli, prompting it to activate bypass metabolic pathways. This may explain the detection of fatty acid-related alkanes, such as dodecane and pentadecane, typical products of fatty acid metabolism. To address this, we plan to improve environmental GPP stability and avoid these bypass pathways. Conducting metabolic flux analysis would help trace GPP’s fate and minimize the formation of these byproducts.

We also identified issues related to the purity of the extractant. Using higher-grade ethyl acetate would address the impurities detected and provide a more suitable logP value for extraction.

With more time and resources, we aim to systematically test the effectiveness of individual proteins along the pathway to identify and address errors. This approach will pinpoint specific proteins that need correction to optimize the entire pathway.

Read about our future plans on the Wetlab-Overview page.