Revision as of 22:36, 30 June 2017

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:

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.

Lab Procedures and Experimental Design

To verify correct expression of the FBP/SBPase protein, a fusion with the red fluorescent protein RFP is planned. So, the construct of the gene to insert goes as follows:

HSP70A/RBCS2-cTP-FBP/SBPase-RFP-NOST

Afterwards, ligation to a plasmid must be made. We are evaluating the possibility of using pCAMBIA1301 or pCAMBIA1304.

Once the insertion has been made, the plasmids must be analyzed in electrophoresis. For this, we must run a sample of ligated plasmid, unligated lasmid, ladder and solution without plasmid. A PCR is also a viable option.

Then, extraction of the resulting bands from the electrophoretic gel shall be done.

Once the plasmids with correct insertion of the gene has been successfully verified, transformation of E. coli will be done to amplify the availibility of the modified vector. To make this possible, LB culture medium must be prepared. Afterwards, electroporation of the bacteria will be conducted to ensure plasmid flux through E. coli, though chemical methods of transformation are also being discussed. Then, the bacteria will be transferred to selective medium in order to distinguish transformed and non-transformed colonies, probably containing kanamycin and Xgal. Once a sufficient amount of E. coli population is achieved, DNA material is then extracted and purified to obtain the desired vector.

Transformation of Chlamydomonas reinhardtii

Two types of transformation methods are currently being evaluated: the traditional and low-efficient glass bead method, and the use of Agrobacterium tumefaciens.

About the hindering mechanism of starch synthesis

As told previously, this objective will be achieved through the use of antisense DNA. The genes to be inserted would be:

MetE-antiSTA1-NOST, MetE-antiSTA6-NOST, MetE-antiGBS2-NOST

Experimental procedure for the transformation process is analogous to the recently described one. However, the correct sequence of the gene is still being evaluated.

Mathematical Modelling

In our project, the genetic circuit design is valued by the use of engineering principles. Part of this consists in making a mathematical model capable of describing our circuit by the means of parameters relevant to the system, to be able to improve them before the lab experiences.

With our model a lot of questions concerning the metabolic and genetic process of our project, like: Which enzymes involved in the Calvin cycle have more effect in carbon uptake? How does light affect the kinetics of HSP70A/RBCS2 promoter and its consequences on the mechanisms involving FBP/SBPase? What can we do to quantitatively control glucose availability and avoid its deviation to other metabolic routes? What parameters are necessary to improve microalgae growth and production of glucose? How will we build bioreactors that are compatible with people's aesthetic tendencies (more detail in Human Practices)?<\p>

To make all of this possible, we will focus on resolution of a dynamic system with a great volume of bibliographic data for the establishment of a correct B12 incorporation cycle in order to fully control starch synthesis, and simulation of steady states through the use of metabolic flux analysis in order to predict effectiveness of gene insertions.<\p>

Once these questions are answered, various adjustments will be made so a predictive model is achieved for our system.

Human Practices

Strategy

Investigate how much does Chilean people know about contamination, science, biotechnology, eco-friendly production, public spaces, etc. This info will be obtained through several surveys conducted in Santiago de Chile (capital). In response to the information obtained we will start to empower the people by doing workshops, focus groups, talks, visits to laboratories, etc. and design interesting proposal for them. As a general result of all the surveys, we want to model a couple of bioreactors with friendly shapes for people in order to be emplaced in public spaces such as squares, streets, parks, museums, highway, etc.

Financial Resources and Diffusion

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 <h2> Mathematical Modelling </h2>
-<p> [[bla bla bla]] </p></div>
+<p>In our project, the genetic circuit design is valued by the use of engineering principles. Part of this consists in making a mathematical model capable of describing our circuit by the means of parameters relevant to the system, to be able to improve them before the lab experiences.</p>
+<p>With our model a lot of questions concerning the metabolic and genetic process of our project, like: Which enzymes involved in the Calvin cycle have more effect in carbon uptake? How does light affect the kinetics of HSP70A/RBCS2 promoter and its consequences on the mechanisms involving FBP/SBPase? What can we do to quantitatively control glucose availability and avoid its deviation to other metabolic routes? What parameters are necessary to improve microalgae growth and production of glucose?
+How will we build bioreactors that are compatible with people's aesthetic tendencies (more detail in Human Practices)?<\p>
+<p>To make all of this possible, we will focus on resolution of a dynamic system with a great volume of bibliographic data for the establishment of a correct B12 incorporation cycle in order to fully control starch synthesis, and simulation of steady states through the use of metabolic flux analysis in order to predict effectiveness of gene insertions.<\p>
+<p>Once these questions are answered, various adjustments will be made so a predictive model is achieved for our system.</div>
 <div class="column half_size" >

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