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Greenhardtii Project

Have you ever feel like some things shouldn't be happening? Like absurdly hot days in summer or very short winters, a lot of torrential rains or even extensive droughts. Not to mention heartbreaking sceneries like glacial melting or coral bleaching. The cause to these problems is well known and we cannot ignore this problem: global warming. Earth's beautiful biological diversity is in trouble and decisions must be made to revert this. Staying just where we are and doing nothing while we cling to an everlasting cloud of uncertainty is just going to bring us forth an impending world of demise. When will this end? We don´t know. How can we avoid this? To that we all have an answer, but constant industrial recklessness makes it pretty difficult to actually change anything. What can we do to help? Well, that's what Greenhardtii Project is about: we want to reduce carbon dioxide emissions by the means of the ability of microalgae to capture this problematic gas and use it to produce materials of interest. To make it even better, we will artificially increase its carbon uptake capacity and hinder the natural synthesis of starch to concentrate glucose availability. In other words, we plan to establish a production platform that uses carbon dioxide as raw material.

Overview

The microorganism that we use is Chlamydomonas reinhardtii, a microalgae extensively used for genetic studies and algae metabolism.

In regard to the increase in carbon uptake capacity, we focus in the Calvin cycle and consider the possibility of an additional enzyme to accelerate the process. Specifically, we evaluated the expected effects of incorporating FBP/SBPase to this metabolic route. Moreover, we ran short simulations on the kinetics of each reaction involved in the cycle and the consequences of modifying the parameters of the enzyme involved in them. We then came to the conclusion that adding FBP/SBPase is feasible. To allow a constant genetic expression of the FBP/SBPase gene we decided to use a strongly constitutive (but enhanceable with light and temperature) fusion promoter HSP70A/RBCS2, along with the NOST terminator and a chloroplast peptide signal (cTP).

To hinder the synthesis route of starch, we plan to control the deactivation of genes STA1 and STA2 to prevent expression of Glucose-1-phosphate adenylyltransferase enzyme that catalyzes the conversion of Glucose-1-phosphate to ADP-glucose, a compound prior to amylose (which is the precursor of starch), and the deactivation of gene GBS2 that encodes the enzyme Granule Bound Starch Synthase, avoiding the transformation of Glucose-1-phosphate to UDP-glucose. To do such regulation we want to incorporate antisense DNA, that way the RNA transcribed will be complimentary to the RNA of the original genes, causing them to bind and block (partially or completely) the translation process. The promoters used for these will be the B12-responsive MetE. Even though it is known that the use of vitamin B12 for a constant regulation of a microalgae culture is not economically feasible, its use will be for evaluations of the genetic circuit only. In a further development of the project we will change to a different promoter, possibly a light-induceable one. The terminator will also be NOST.

Here we present an image that summarizes our genetic circuit:

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As you can see, a product has not yet been defined to actually verify successful functionality of the project. This is still in discussion and has been quite a headache so far. The project not only consists in the design of our genetic circuit, but it also focuses on a correct transformation of Chlamydomonas reinhardtii by an exhaustive experimental procedure, a mathematical model that told us what enzyme is preferable to add and in a further development it will establish other parameters to increase cellular concentration and glucose availability, a human practice that will inform us about real population tendencies in order to design attractive bioreactors, and a financial strategy to obtain necessary money to attend the Giant Jamboree and make Greenhardtii Project possible.

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OpenBio - University of Chile 2017

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