Team:Lethbridge/Plant
Plant Synthetic Biology
Overview
We have contributed:
- Evidence that the acidophilic Cyanidioschyzon merolae microalgae strain is stable in the acidic stomach environment and thus is an optimal vehicle for oral delivery of protein drugs to the gut.
- New parts that are codon-optimized for microalgae, specifically the model Chlamydomonas reinhardtii chassis.
- Microalgae growth and final product preparation protocols.
- Future considerations to improve biosafety.
Cyanidioschyzon merolae chassis
As far as we know, we are the first iGEM team to work with C. merolae as a chassis.
The 2016 Cambridge iGEM team provided many microalgae parts to encourage the use of C. reinhardtii as a model microalgae chassis and listed numerous benefits of using microalgae over other expression systems (summarized below). However, we found that C. merolae, an acidophile, addresses some of the remaining disadvantages of using microalgae: it grows readily in acidic environments, which can be exploited to ensure culture sterility, and the cell wall is resistant to degradation in the stomach, making it a superior vehicle for oral drug delivery to the intestines.
| Post-translational modifications |  |
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| Proteins are not expressed as insoluble aggregates | |
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| Good expression yields | |
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Does not require complex growth media or conditions | |
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| Resilient to contamination |  |
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| Fast growth | |
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| Edible drug delivery possible | |
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Drug Delivery Capabilities
Based on our acid degradation tests (refer to our Results page), we confirmed that the cell wall of the acidophilic C. merolae chassis, unlike other microalgae strains, could withstand the harsh acidic conditions of the stomach to enable drug delivery to the gut. Below is a comparative summary of the results from three tested microalgae strains:
Microalgae Parts
We developed several codon-optimized composite parts for genomic integration and testing in the model C. reinhardtii chassis including constructs containing either the single-chain insulin, SCI-57, or proinsulin peptides (refer to Parts page). Both of these insulins were chosen on the basis that they would most likely mimic native receptor binding activity and endogenous insulin function. Each of these insulin parts also included: a histidine tag for protein extraction, furin cut sites to aid proinsulin processing and ensure appropriate protein folding, flexible linkers to reduce steric hindrance, a cell-penetrating peptide to ensure uptake by gut epithelial cells, and a fused RFP (red fluorescent protein) for ratiometric quantification of insulin production.
For cloning in C. reinhardtii, we used the pSRSapI vector, which can be used to target and integrate our construct within chloroplasts (Wannathong et al., 2016). This vector enables:
- Targeting of the expression cassette to a neutral locus downstream of psbH by homologous recombination.
- Simple phototrophic selection for restoration of the endogenous gene psbH as an effective but benign selectable marker in cell wall-deficient C. reinhardtii strains.
- Fusion of the transgenic coding sequence behind the promoter and 5′ UTR element of the highly expressed endogenous gene, psaA.
- Avoidance of gene-silencing mechanisms.
Microalgae Growth & Preparation Protocols
As the first team to work with the C. merolae chassis, we have provided many useful protocols for the laboratory-based growth of this unique strain. In addition, because the shelf life of injectable insulin is only one month and requires refrigeration, we also tested three alternative storage options for Algulin: lyophilization, solar or heat-based dehydration, and freezing in glycerol. Please refer to our Experiments page and our Results page for more information.
Future Directions - Biosafety
Light Antenna
We have developed a NAB1 constitutive expression construct (BBa_K3237024) and an antisense NAB1 Dicer-like mediated repressor construct (BBa_K3237012) to manipulate the light antennas of the microalgae. In wildtype microalgae in high light conditions, NAB1 sequesters light antenna mRNA and prevents its translation, truncating the light antenna to prevent photobleaching. By integrating a consitutively expressed, antisense NAB1 repressor system into our C. merolae chassis, we not only enable improved growth efficiency and consistency in a low light lab environment, but would also vastly reduce environmental competitiveness in the wild because the modified microalgal cells would be incapable of modifying their light antennas to adapt to higher light environments and would die from photobleaching.
Kill Switch
To ensure our genetically modified C. merolae strain cannot accidentally contaminate wild populations, we are also planning to implement a kill switch. Celesnik et al. (2016) used expression of the non-specific nuclease, NucA from Anabaena, under a metal-ion inducible promoter to induce conditional cell lethality from DNA degradation. We intend to use a similar system except we will use a zinc-inducible constitutive promoter to induce expression of NuiA, a NucA repressor, to limit NucA expression in microalgae grown in zinc-supplemented media. In the wild, it is unlikely that sufficient zinc will be present for expression of NuiA and repression of NucA will be lifted, enabling DNA degradation and cell death. This system remains to be tested to ensure that sufficient cell lethality is achieved to be an effective biosafety measure.
References
Celesnik, H., Tansek, A., Tahirovic, A., Vizintin, A., Mustar, J., Vidmar, V., & Dolinar, M. (2016). Biosafety of biotechnologically important microalgae: Intrinsic suicide switch implementation in cyanobacterium Synechocystis sp. PCC 6803. Biology Open, 5, 519-528.
Wannathong, T., Waterhouse, J.C., Young, R.E., Economou, C.K., & Purton, S. (2016). New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii. Appl Microbiol Biotechnol, 100, 5467-77.