The plasmids we worked with were the pJBEI-6410 and pJBEI-6411, ordered from AddGene.
We used a double-transformation to transform both plasmids into chemically competent E. coli BL21 and DH5α cells, supplied by Dan White of Sagona Lab and also from Dr. Deborah Brotherton.
Below is the list of enzymes of interest that pJBEI-6410 codes for.
Below is the list of enzymes that allow the conversion of limonene into perillyl alcohol.
Monix001 is our construct that is composed of the CpxP (BBa_K4600003), TetR (BBa_K4600002) and AcrB pump sequence (BBa_K4600001), submitted as BBa_K4600006 on the iGEM part's registry.
Monix001 is inserted into pJBEI-6411 as a linear construct, and functions when produced perillyl alcohol causes membrane stress which is recognised by the CpxP region. This natively promotes the expression of downstream TetR and AcrB sequences.
Tet sequences are found in E.coli to pump out tetracycline antibiotic. TetR acts as dimers to transcriptionally repress (or activate) the expression of the attached gene of interest. The promoter region will be placed upstream of the promoter for our specific enzymes. Tetracycline (Tc) or tetracycline-derivatives (e.g. dox or aTC) will bind to the TetR to cause a conformational change and unbind it from the tetO elements - reactivating transcription of our enzymes.
In the context of our system this means that when the stress protein Cpx-R-P upregulates TetR production, TetR will bind at the pTet region, and suppress the promoter of the genes encoding mevalonate enzymes responsible for creating the toxic products.
We made both pJBEI-6410 and pJBEI-6411 tet inducible. Firstly, the TetR region on our construct is inserted into pJBEI-6411, making pJBEI-6411*. This was followed by adding a pTet region, (BBa_R0040), into pJBEI-6410, making pJBEI-6410*.
This feedback represses expression of the listed enzymes and therefore prevents acetyl co-enzyme A from being transformed in the mevalonate pathway and so will not produce the toxic intermediates. This allows the cell to recuperate and repair membrane damage, as no perillyl alcohol is being produced.
Alongside this, the AcrB pump is also promoted. This allows for the existing limonene and perillyl alcohol to be transported out of the cell to prevent any further damage to the cell's membrane and therefore the cell's survival.
Due to the limited time frame we could not fully implement our idea and see it come to fruition as a viable alternative to current perillyl alcohol production via lavender harvesting. If we could continue our project, we would focus on further modifying the AcrB efflux pump to be more specific to our key product we want to expel - perillyl alcohol. This could be done via directed mutation, by growing a series of colonies of E. coli strains containing the AcrB gene, and selecting the colonies with the highest growth in perillyl alcohol solution. We hope to see a mutation on the target sites of the pump, making it more specific to perillyl alcohol (Sharma et al., 2019)
We can also look at the protein sequence of the pump in order to analyse the target site where the substrate binds, and do a series of site directed mutagenesis. We hope to increase the amount of Perillyl Alcohol being removed, while also reducing the amount of perillyl alcohol reverting back to limonene which can be even more toxic to the cell. This achieves a dual benefit where the production of perillyl alcohol is enhanced as it is pumped out and easily purifiable, while also alleviating cellular toxicity (Dunlop et al., 2011).
When ordering and quality checking our pJBEI-6410 plasmid, we ran into some troubling restriction digest gels. Despite repeated attempts and with another restriction site (HindIII), we still did not see expected results.
And so we started on a long back and forth troubleshooting process with AddGene while we continued on with our sample, despite some concerns. It is important to note that despite the odd results, the pJBEI-6410 plasmid still worked in tandem with pJBEI-6411 to produce perillyl alcohol as seen in our results.
Eventually AddGene decided to sequence their own stock, with the same restriction site's we'd used and provided us with an update.
AddGene also had peculiar results with their EcoRI and SacI testing, and after double checking their FASTQ files, the sequence seemed to be of good quality. Therefore, they suggested that as the plasmid has several repetitive elements and it is quite large, so it may be somewhat unstable. If so, it may be particularly important to screen several colonies of this plasmid in order to isolate the full, intact plasmid.
As we were under time pressure, we instead decided to sequence our plasmid, with the Plasmidsaurus company, and when compared with AddGene's sequence of the plasmid it all seemed to be OK. Therefore, more than likely there was an issue when reviving the cells from the stab we'd received as it was quite a large plasmid. Despite this, our plasmid operated perfectly for our uses but we thought it best to mention this slight discrepancy.
This year Team BioMonix participated in the InterLab study, specifically on Experiment 1
As part of the calibration process, we performed serial dilutions with various fluorescent substances, including green fluorescent, sulforhodamine 101 (red) fluorescence, Cascade Blue (blue) fluorescence, and the absorption measurements of NanoCym 950nm monodisperse silica nanoparticles.
In Experiment 1, we have measured the fluorescence emitted by various bacteria containing one or more fluorescent proteins. To facilitate this, we have prepared competent cells, specifically DH5α. Subsequently, we carried out the necessary incubation steps and conducted precise OD (Optical Density) measurements.
Our data collection, however, presented some anomalies, with certain data points not adhering to the expected patterns. This prompted us to delve deeper into the intricacies of competent cell preparation, emphasising the criticality of contamination reduction. Additionally, we tried different protocols to determine the root of the anomalous results. Having discussed with other iGEM teams that also participated in the InterLab study we found that anomalous data was common. Therefore, we uploaded our results making sure to mention this find in our description.
1. Sharma, A., Gupta, V. K., & Pathania, R. (2019). Efflux pump inhibitors for bacterial pathogens: From bench to bedside. Indian Journal of Medical Research, 149(2), 129.
2. Dunlop, M. J., Dossani, Z. Y., Szmidt, H. L., Chu, H. C., Lee, T. S., Keasling, J. D., Hadi, M. Z., & Mukhopadhyay, A. (2011a). Engineering microbial biofuel tolerance and export using efflux pumps. Molecular Systems Biology, 7(1), 487.