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One construct aims to express MlrA on the outer membrane of E. coli. The advantage of this system is that microcystin does not have to be transported into the cytoplasm for degradation, but can be broken down directly within proximity of E. coli. We expressed MlrA on the outer membrane via a fusion protein, consisting of the MlrA linked with PgsA (poly-γ-glutamate synthetase A), a protein natively found in Bacillus subtilis (Narita et al., 2006). Though past iGEM teams, such as JNU-China from 2019, genetically engineered Corynebacterium glutamicium to produce PgsA for various applications such as food additives, anti-freeze, and protective antigen coupling, PgsA can also act as an anchoring motif. This protein and has been used to heterologously express proteins such as α-amylase, lipase B, Laccase CotA, and VP2—an antigen for avibirnavirus in chickens (Narita et al., 2006; Zhang et al., 2018; Maqsood et al., 2018). Therefore, while anchoring MlrA with PgsA is novel, it is derived from a methodology shown to work in other proteins. Ultimately, the novel BioBrick we create can be used by future teams to improve and expand upon our research.
The second construct aims to secrete MlrA into the periplasm, where the enzyme can interact with MC-LR that freely diffuses through the outer membrane. The bacterial twin arginine translocase (Tat) pathway transports fully folded proteins across the inner membrane to the periplasmic space, thereby offering a promising solution to the problems posed by heterologous expression of MlrA in E. coli (Lee et al., 2006).
Despite the potential of the Tat secretion system, one drawback that limits industrial applications is the relatively low yield of exported heterologous proteins. This low yield is primarily due to the small amount of Tat secretion machinery naturally produced in E. coli, as few native proteins proteins utilize the pathway (Barrett et al., 2003). Our team hoped to address this limitation by reproducing the super-secreting “TatExpress” strain of E. coli engineered by Browning et al. The strong IPTG inducible promoter Ptac was introduced upstream of the TatABCD operon on the chromosome, producing a strain with enhanced secretion capacity while avoiding the negative selective pressures introduced by the use of multiple plasmids bearing an extra copy of the gene with the strong promoter (Lee et al., 2006; Browning et al., 2019). The TatExpress strain was subsequently transformed to secrete MlrA. The plasmid insert contained MlrA (Part:BBa_K1907002) fused with the N-terminal TorA signal peptide described by Browning to target the protein to the Tat machinery, and a C-terminal 6xHIS tag for affinity chromatography. MlrA was under the control of a pTet inducible promoter (Part:BBa_K3171173), enabling us to separately induce and repress TatABCD and MlrA independent of each other. TatABCD was induced first while MlrA was repressed to allow cells to produce the secretion machinery, and afterwards TatABCD was repressed and MlrA induced. This design allowed us to minimize the energetic costs to the cells by either expressing Tat machinery or MlrA, but not both simultaneously.
Cornell’s 2019 iGEM Team achieved some success degrading MC-LR using E. coli that heterologously secreted MlrA to the periplasm with the Tat pathway. We hope our TatExpress colonies and dually inducible system will be an improvement upon their work.
Novel Detection System:
Two-hybrid assays are a popular molecular biology technique used to screen protein-protein interactions. Within the most popularly characterized yeast two-hybrid system (Y2H), a transcription factor is split into two components, DNA- binding domain (DBD) and activating domain (AD). The two proteins of interest (bait and prey) are respectively bound to these domains. Interaction between the DBD and AD reconstitutes the transcription factor, which can then bind to the upstream activation sequence, ultimately activating the transcription of a reporter gene (Brückner et al., 2009). Though the two-hybrid assay is most notably used in yeast, there have been other variations used in protein interactions, such as the bacterial two-hybrid assay, one hybrid, and three-hybrid variants (Karimova et al., 2008).
A particularly significant alternative to the Y2H utilizes a bacterial chassis and is known as the bacterial adenylyl cyclase two-hybrid (BACTH) system. Whereas the Y2H relies on transcription factors, BACTH is dependent on the reconstitution of adenylyl cyclase (AC), the enzyme that synthesizes cyclic AMP (cAMP) (Moore et al., 2016). Within Bordella pertussis, the strain where the BACTH assay was first studied, it was found that the catalytic domain of adenylyl cyclase could be divided into two subdomains: T18 and T25. In the presence of calmodulin, these subdomains could come together and stimulate cAMP production. In the BACTH, proteins of interest are attached to these domains and the interactions cause cAMP to be produced. From here, cAMP binds to catabolite activator protein (CAP) to create the CAP/cAMP complex, which then functions to activate transcription of reporter genes (Battesti & Bouveret, 2012).
Another base component of the adapted BACTH system assay is a protein phosphatase assay. Microcystin's natural ability to inhibit protein phosphatases 1 and 2a (PP1 or PP2A) is taken advantage of to create a colorimetric detection method. The substrate p-nitrophenyl phosphate is acted upon by either PP1 or PP2A to produce a yellow color that can be quantified through spectrophotometry (Akin-Oriola & Lawton, 2010).
In this adapted BACTH assay, the proteins of interest include PP1, previously introduced in the discussion of the pNPP assay, as well as glutathione (GSH), an antioxidant. Both PP1 and GSH bind to MC-LR; PP1 is inhibited by MC-LR’s Adda site, whereas GSH is able to reduce the overall toxicity of MC-LR. These three components are able to form a complex, and when PP1 and GSH are bound to T18 and T25 respectively, they cause the two subdomains to come close enough together to reconstitute adenylyl cyclase and stimulate cAMP production once again (Zong et al., 2017).
A variation of this proposed idea was successfully attempted by the iGEM 2009 Tianjin team, with the difference of using a yeast two-hybrid assay instead. Our proposed idea involves a novel method for MC-LR detection, which has the potential to combine both detection and degradation aspects for future projects/teams.
Akin-Oriola, G., & Lawton, L. (2010). Detection and quantification of toxins in cultures of microcystis aeruginosa (pcc 7820) by hplc and protein phosphatase inhibition assay effect of blending various collectors at bulk. African Journal of Science and Technology, 6(1). https://doi.org/10.4314/ajst.v6i1.55157
Barrett, C. M. L., Ray, N., Thomas, J. D., Robinson, C., & Bolhuis, A. (2003).Quantitative export of a reporter protein, GFP, by the twin-arginine translocation pathway in Escherichia coli. Biochemical and Biophysical Research Communications, 304(2), 279–284. https://doi.org/doi.org/10.1016/s0006-291x(03)00583-7
Battesti, A., & Bouveret, E. (2012). The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. Methods, 58(4), 325–334. https://doi.org/10.1016/j.ymeth.2012.07.018
Browning, D. F., Richards, K. L., Peswani, A. R., Roobol, J., Busby, S. J. W., & Robinson, C. (2017). Escherichia coli “TatExpress” strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway. Biotechnology and Bioengineering, 114(12), 2828–2836. https://doi.org/doi.org/10.1002/bit.26434
Brückner, A., Polge, C., Lentze, N., Auerbach, D., & Schlattner, U. (2009). Yeast Two-Hybrid, a Powerful Tool for Systems Biology. International Journal of Molecular Sciences, 10(6), 2763–2788. https://doi.org/10.3390/ijms10062763
Dziga, D., Wladyka, B., Zielińska, G., Meriluoto, J., & Wasylewski, M. (2012). Heterologous expression and characterisation of microcystinase. Toxicon, 59(5), 578–586. https://doi.org/doi.org/10.1016/j.toxicon.2012.01.001
Karimova, G., Dautin, N., & Ladant, D. (2008). Interaction Network among Escherichia coli Membrane Proteins Involved in Cell Division as Revealed by Bacterial Two-Hybrid Analysis. Journal of Bacteriology, 190(24), 8248. https://doi.org/10.1128/jb.01470-08
Lee, P. A., Tullman-Ercek, D., & Georgiou, G. (2006). The Bacterial Twin-Arginine Translocation Pathway. Annual Review of Microbiology, 60(1), 373–395. https://doi.org/doi.org/10.1146/annurev.micro.60.080805.142212
Moore, C., Juan, J., Lin, Y., Gaskill, C., & Puschner, B. (2016). Comparison of Protein Phosphatase Inhibition Assay with LC-MS/MS for Diagnosis of Microcystin Toxicosis in Veterinary Cases. Marine Drugs, 14(3), 54. https://doi.org/10.3390/md14030054
Maqsood, I., Shi, W., Wang, L., Wang, X., Han, B., Zhao, H., Nadeem, A. M., Moshin, B. S., Saima, K., Jamal, S. S., Din, M. F., Xu, Y., Tang, L., & Li, Y. (2018). Immunogenicity and Protective efficacy of orally administered recombinant Lactobacillus Plantarum expressing VP2 protein against IBDV in chicken. Journal of Applied Microbiology, 125(6), 1670–1681. https://doi.org/10.1111/jam.14073
Maseda, H., Shimizu, K., Doi, Y., Inamori, Y., Utsumi, M., Sugiura, N., & Kobayashi, M. I. (2012). MlrA located in the inner membrane is essential for Initial degradation Of microcystin In sphingopyxis sp. C－1. Japanese Journal of Water Treatment Biology, 48(3), 99–107. https://doi.org/10.2521/jswtb.48.99
Narita, J., Okano, K., Tateno, T., Tanino, T., Sewaki, T., Sung, M.-H., Fukuda, H., & Kondo, A. (2005). Display of active enzymes on the cell surface of Escherichia coli using PgsA anchor protein and their application to bioconversion. Applied Microbiology and Biotechnology, 70(5), 564–572. https://doi.org/10.1007/s00253-005-0111-x
Zhang, Y., Dong, W., Lv, Z., Liu, J., Zhang, W., Zhou, J., Xin, F., Ma, J., & Jiang, M. (2018). Surface display of bacterial Laccase CotA on Escherichia coli cells and its application in Industrial Dye Decolorization. Molecular Biotechnology, 60(9), 681–689. https://doi.org/10.1007/s12033-018-0103-6
Zong, W., Wang, X., Du, Y., Zhang, S., Zhang, Y., & Teng, Y. (2017). Molecular Mechanism for the Regulation of Microcystin Toxicity to Protein Phosphatase 1 by Glutathione Conjugation Pathway. BioMed research international, 2017, 9676504. https://doi.org/10.1155/2017/9676504
Toxins from cyanobacterial harmful algal blooms (cHABs) contaminate lakes and cause severe illness or even death in humans and animals who consume contaminated water. Microcystin-LR (MC-LR) is one such toxin that is ubiquitous in lakes with cHABs. MC-LR is a potent hepatotoxin and carcinogen, damaging the liver by binding to protein phosphatases and inhibiting key biochemical pathways (Woolbright et al., 2017). This not only creates a significant threat to human and animal health, but also leads to a biodiversity crisis (Magrann et al., 2012). The severe overgrowths of aerobic algae that occur during cHABs also consume excessive amounts of oxygen, decreasing the oxygen saturation of the body of water. Oftentimes, the resultant oxygen saturation level is so low that it is prohibitive to the survival of other aerobic species of algae, zooplankton, plants, and animals in the aquatic biome. In recent years, algal blooms have become an increasingly concerning public health, environmental, and biodiversity crisis.
Current industrial Methods
- Activated Charcoal Absorption
Global warming and climate change have exacerbated the issue. In recent decades, there has been a dramatic increase in the incidence of cHABs worldwide, attributable not only to a greater frequency of preexisting blooms but also to the spread of toxic species to previously unaffected areas (Anderson et al., 2021). Ocean warming, fertilizer runoff, and aquaculture expansion are all factors that exacerbate the issue. Among the hundreds of different cHAB toxins, MC-LR levels specifically have been found to positively correlate with water temperatures (Lürling et al., 2017). With no end in sight for these exacerbating factors, cHABs will only continue to become an increasingly severe concern; the development of an affordable and effective method of MC-LR degradation is crucial.
The Solution: Introducing Pac-Coli
Our project aims to develop a novel microcystin degradation solution by utilizing recombinant E. coli that heterologously over-express MlrA, or microcystinase, an enzyme known to degrade MC-LR in the native Sphingomonas sp. Two approaches have been developed to address this issue. One strain will express MlrA anchored to the outer membrane via a fusion protein with poly-γ-glutamic (PgsA), and the other will freely secrete MlrA into the periplasm via a modified twin arginine translocase (Tat) secretion system. This project also proposes a microcystin detection method using a two-hybrid bacterial assay, which generates a fluorescence signal based on protein-protein interactions through the use of the messenger molecule cAMP.
Anderson, D. M., Fensin, E., Gobler, C. J., Hoeglund, A. E., Hubbard, K. A., Kulis, D. M., Landsberg, J. H., Lefebvre, K. A., Provoost, P., Richlen, M. L., Smith, J. L., Solow, A. R., & Trainer, V. L. (2021). Marine harmful algal blooms (HABs) in the United States: History, current status and future trends. Harmful Algae, 102, 101975. https://doi.org/10.1016/j.hal.2021.101975
Gobler Lab Joins the Battle Against Algal Blooms | | SBU News. (2021, April 26). SBU News. https://news.stonybrook.edu/university/gobler-lab-joins-the-battle-against-algal-blooms/
Lürling, M., van Oosterhout, F., & Faassen, E. (2017). Eutrophication and Warming Boost Cyanobacterial Biomass and Microcystins. Toxins, 9(2), 64. https://doi.org/10.3390/toxins9020064
Magrann, T., Dunbar, S. G., Boskovic, D. S., & Hayes, W. K. (2012). Impacts of Microcystis on algal biodiversity and use of new technology to remove Microcystis and dissolved nutrients. Lakes & Reservoirs: Research & Management, 17(3), 231–239. https://doi.org/10.1111/lre.12000
Woolbright, B. L., Williams, C. D., Ni, H., Kumer, S. C., Schmitt, T., Kane, B., & Jaeschke, H. (2017). Microcystin-LR induced liver injury in mice and in primary human hepatocytes is caused by oncotic necrosis. Toxicon, 125, 99–109. https://doi.org/10.1016/j.toxicon.2016.11.254