Team:USP UNIFESP-Brazil/Plant

AlgAranha Team USP_UNIFESP-Brazil

iGEM 2016

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Plant Synthetic Biology

Chlamydomonas reinhardtii is an important non-vascular plant used as a model organism for studying a diverse number of organisms’ cell functional systems, life cycles and physiological processes. This microalga was first known as a great model to study photosynthesis and biogenesis of chloroplast, when some studies showed that a mutant without chlorophyll was able to grow in cultivation media without acetate. Later, some researchers continued studying gene regulation to identify nuclear genes that regulate the expression of genes encoded in the chloroplast in photosynthetic organisms (Merchant et al.; Mayfield and Franklin; Gimpel et al.). Phototaxy and flagella are related and investigated to help unveiling changes in flagellar beating and male fertility (Pazour et al.). Other great researches are about its sexual cycle with stable and viable mating types that enables better and fast control of this species life cycle; and finally, C. reinhardtii is considered the best organism to study circadian rhythms (Harris; Mittag, Kiaulehn and Johnson). The circadian clock system is an endogenous biological program that controls events like, metabolic, physiological and behavioral ones, so they occur in the optimal daily cycle phase. C. reinhardtii is the preferred organism for these studies, also because of the great amount of genetic and molecular techniques already developed based on this microalga fully sequenced genomes (mitochondrial, chloroplast and nucleus). Other related advantages are its simple life-cycle and ease to isolate mutants (Specht, Miyake-Stoner and Mayfield). As Chlamydomonas reinhardtii is a model organism for a vast range of studies, all the protocols, tools and parts made for this plant chassis will be useful for other IGEM teams.

As mentioned at the beginning, C. reinhardtii is a non-vascular plant, i.e., it is an organism that is easily scaled as it grows in liquid cultivation media and it has fast doubling time, unlike plants (Mishler). These considerations indicate this organism could be a great production platform, not only for native products, but also recombinant protein production (Specht, Miyake-Stoner and Mayfield).

This microalga chloroplast protein expression platform has demonstrated its utility in the production of proteins such as malaria vaccines and cancer immunotoxins (Gregory et al.; Tran et al.). The chloroplast is a better expression platform when compared to the nucleus, due to its transformation, which occurs through homologous recombination. Some studies demonstrated also high heterologous protein accumulation in the chloroplast when using a psbA promoter and 5’UTR, only in psbA knockout strain (Manuell et al.; Rasala, Muto, et al.). In the nucleus, instead, the heterologous gene transformation occurs randomly, there is gene silencing and promoters and vectors are still being developed, which shows a need. There are some common used promoters from HSP70A, psaD, rbcS2 genes (Schroda, Beck and Vallon; Fischer and Rochaix), but better expression is obtained when chimeric promoters are used or intron sequences are placed in between (Schroda, Blocker and Beck). To improve expression and overcome silencing effects in C. reinhardtii nucleus, we used the vector developed by (Rasala, Lee, et al.), but expressing the fluorescent protein mcherry. This impressive part is named BBa_K2136010 (5’ cassette for Chlamydomonas reinhardtii transgenic expression), it is a vector that contain the promoter hsp70A/rbcs2, followed by the bleomycin/zeocin resistance gene and the self-cleaving 2A peptide sequence (FMDV/Foot-and-mouth-disepse-virus) to overcome gene silencing. (Rasala, Lee, et al.). Figure 1: Construct for the recombinant gene expression in the nuclear genome from Chlamydomonas reinhardtii

Chlamydomonas reinhardtii nucleus as an expression system present some characteristics as advantages when compared to the chloroplast, for example: inducible gene expression, protein localization to subcellular locations, and eukaryotic post-translation modification including glycosylation. One of the main special attributes of this microalga is the secretion ability, which is important for purification. The secreted heterologous protein is successfully demonstrated on the figure 2. Figure 2: Laser passing through cellular supernatant. A - Laser is passing through a wild type C. reinhardtii supernatant. B- Laser is passing through a transformed C. reinhardtii producing mCherry.

Since this is a good expression system and one of the difficulties to create a useful recombinant algae has been the lack of genetic tools to be able to obtain better heterologous protein expression in the nucleus, in our project we used the vector described by (Rasala, Lee, et al.), to make the improvement by cloning the fluorescent reporter mcherry gene as a genetic tool. Fluorescent proteins are intrinsically used for two purposes: in combination with promoters to characterize the expression on a gene of interest, and also as tags to be able to visualize expression and the protein of interest localization (Hutter). GFP (Green Flurescent Protein) was the fluorescent protein that marked the new era of fluorescent reporters and since then, there has been made a lot of improvements in GFP to increase its brightness and increase color variants, but also, other fluorescent proteins were isolated from different organisms. Mcherry gene is isolated from Discosoma sp. and its encoded protein is usually chosen for genetic studies due to its red color, small size, photostability if compared to the other monomeric fluorophores and quick maturation, which allows it to be visualized quicker after translation (Shaner et al.).

Considering all these advantages and that, there is a lot to be done to deliver a better expression system to the scientific community and society, we selected Chlamydomonas reinhardtii as our chassis and improved it as a genetic tool. We would like to be evaluated for this Best Advancement in Plant Synthetic Biology, because we showed that the chosen chassis can express the fluorescent protein mCherry, and can be used as a vector to express proteins of interest that will be easily and quickly detected for unlimited range of studies in vitro and in vivo. Figure 3: This plate shows the successful Chlamydomonas reinhardtii transformation with the vector shown on the Figure 1, containing mcherry as the gene of interest and selected with the antibiotic zeocin (ble selection). Figure 4: This figure shows the mcherry fluorescence of the best 5 recombinant Chlamydomonas reinhardtii expressors (A1, A2, A6, A7, B1) from the samples collected every 12h in a cultivation during 96h. Please check here for more experimental data: http://parts.igem.org/Part:BBa_K2136016

Fischer, N., and J. D. Rochaix. "The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii." Mol Genet Genomics 265.5 (2001): 888-94.

Gimpel, J. A., et al. "Production of recombinant proteins in microalgae at pilot greenhouse scale." Biotechnol Bioeng 112.2 (2015): 339-45.

Gregory, J. A., et al. "Alga-Produced Cholera Toxin-Pfs25 Fusion Proteins as Oral Vaccines." Applied and Environmental Microbiology 79.13 (2013): 3917-25.

Harris, E. H. "Chlamydomonas as a Model Organism." Annu Rev Plant Physiol Plant Mol Biol 52 (2001): 363-406.

Hutter, H. "Fluorescent reporter methods." Methods Mol Biol 351 (2006): 155-73.

Manuell, A. L., et al. "Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast." Plant Biotechnology Journal 5.3 (2007): 402-12.

Mayfield, S. P., and S. E. Franklin. "Expression of human antibodies in eukaryotic micro-algae." Vaccine 23.15 (2005): 1828-32.

Merchant, S. S., et al. "The Chlamydomonas genome reveals the evolution of key animal and plant functions." Science 318.5848 (2007): 245-50.

Mishler, B. D. "Deep phylogenetic relationships among "plants" and their implications for classification." Taxon 49.4 (2000): 661-83.

Mittag, M., S. Kiaulehn, and C. H. Johnson. "The circadian clock in Chlamydomonas reinhardtii. What is it for? What is it similar to?" Plant Physiol 137.2 (2005): 399-409.

Pazour, G. J., et al. "Identification of predicted human outer dynein arm genes: candidates for primary ciliary dyskinesia genes." J Med Genet 43.1 (2006): 62-73.

Rasala, B. A., et al. "Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide." PLoS One 7.8 (2012): e43349.

Rasala, B. A., et al. "Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5 ' untranslated region optimization." Plant Biotechnology Journal 9.6 (2011): 674-83.

Schroda, M., C. F. Beck, and O. Vallon. "Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas." Plant Journal 31.4 (2002): 445-55.

Schroda, M., D. Blocker, and C. F. Beck. "The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas." Plant J 21.2 (2000): 121-31.

Shaner, N. C., et al. "Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein." Nat Biotechnol 22.12 (2004): 1567-72.

Specht, E., S. Miyake-Stoner, and S. Mayfield. "Micro-algae come of age as a platform for recombinant protein production." Biotechnol Lett 32.10 (2010): 1373-83.

Tran, M., et al. "Production of Anti-Cancer Immunotoxins in Algae: Ribosome Inactivating Proteins as Fusion Partners." Biotechnology and Bioengineering 110.11 (2013): 2826-35.