Difference between revisions of "Team:USP UNIFESP-Brazil/Plant"

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.).