Team:Bordeaux/Template:OverviewOurSolution
This year, the team is working on an environmental project, trying to remedy problems associated with the use of copper sulfate by bringing new solutions thanks to synthetic biology. To do that, all the work will be done using non-pathogenic micro-organisms which can easily be grown in our lab: Escherichia coli and Saccharomyces cerevisiae. Therefore our research tries to uncover ways to increase the production of this natural product that may decrease the cost in its industrial production for example.
Our project this year is to produce curdlan, a beta 1,3 glucan. Some (1→3)-β-D-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. (CURDLAN + BACTERIA + IMMUNE S) One of the (1→3)-β-D-glucans is call Curdlan, it is produced as a capsule on Agrobacterium sp. ATTC 31749. the capsule formation is correlated with cell aggregation (floc formation). Capsule formation occurs when the N source in minimal medium is depleted, suggesting that the capsule and floc formation together function as protective structures in nutritional stress. Curdlan is typical of a secondary metabolite in that its biosynthesis occurs in the post-stationary growth phase during conditions of N-starvation. The optimisation of curdlan yield thus depends on the formation of a cell mass in the pre-stationary phase and on the biosynthetic capability of cells in the post-stationary phase. and is used as an Extracellular PolySaccharides (EPS). Curdlan is a linear homopolymer of the monosaccharide D-glucose molecules and may have as many as 12,000 glucose units. It is a neutral polymer produced by Alcaligenes faecalis var. myxogenes 10C3 strain. Subsequent research on curdlan production focused on selection of bacterial strains and optimization of the culture media. But we would like to produce Curdlan in another bacteria, E.coli, because we are SWAG (Secretly we adore Glucan) <3 Before starting the project, we took a few weeks to decide which host organism we would use and how they coule be useful. To begin with we looked at three different organisms: Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae. We rapidly eliminated bacillus subtilis from our possible hosts due to it's lack of enzymes involved in the metabolic pathway of beta 1,3 glucans. However, we found that yeast naturally produces curdlan and that E. coli is only missing one enzyme to synthethize curdlan. We therefore concluded that we could keep these two organisms: one where we would overexpress the beta 1,3 glucan prodution using a constititive promoter and one where we would insert the ability to create curdlan by adding the enzyme that is needed. In bacteria, three genes are totally required for curdlan production. These have been identified in Agrobacterium sp. ATCC31749, which produces curdlan in large amounts. This putative operon contains crdS, encoding β-(1,3)-glucan synthase catalytic subunit, flanked by two additional genes : crdA and crdC. The first assists translocation of nascent polymer across cytoplasmic membrane and the second assists passage of nascent polymer across the periplasm. The protective effects are due to the fact that Curdlan forms a capsule that completely surrounds the outer cell surface of bacteria. Now, we are going to put these three genes into Escherichia coli through the intermediary of plasmids and then do a bacteria culture for curdlan production during its stationary phase. Then we will collect only curdlan by a purification method. Finally, curdlan will be sulfated to be in its active form. This sulfation will be made chemically. What is curdlan? How is it naturally synthethised? Why is it useful for bacteria? How are we going to produce it? Can we really call it eco-friendly? What organisms are we going to use? Why? What are the advantages and disadvantages of both organisms? How did we choose the genes? Promoters? How are we going to make the biobrick? What is our biobrick? Yeast cell walls are made up of various layers which are represented in the following diagram. First there is a layer of chitin, then a layer of beta glucans and finally a mixed layer of proteins and mannan. Commonly, the yeast cell wall is made of 5-10% of beta 1,6 glucans and 50-55% of beta 1,3 glucans and beta 1,6 glucans. Since the layer of mannan and proteins as well as chitin is insoluble in alkali solutions, beta glucans are easily separated from the rest of the yeast cell wall. Therefore, the only alkali soluble components are a mix of beta 1,6 and beta 1,3 glucans. (aimanianda et al 2009) In order to separate the two we plan on using beta 1,6 glucanases in order to obtain a solution of beta 1,3 glucans and therefore our curdlan molecule. We therefore decided to over-express the curdlan metabolic pathway by inserting into yeast an inducible promoter (gal1) for the glucan synthase gene (Fks1) hoping that this would allow the cell to produce curdlan in greater quantities. This would allow us to compare our curdlan production in E. coli to the natural production in an organism and the enhanced production through the addition of a promoter. Biobrick schema Curdlan is a linear beta-1,3-glucan. This molecule is a polymer composed of many units of glucose, a commonly used sugar. It is produced by bacteria such as Agrobacterium, even by yeast.
In fact, this β1,3 glucan stimulates the plant's immune system. Activation of the innate immune system before the invasion of pathogens is a way to improve the resistance of plant against infection and reduce the use of chemicals products. Blabla how work curdlan on plant immune system + Schema
Applied to grapevine plants, sulfated curdlan induces the accumulation of phytoalexins and expression of a set of Pathogenesis-Related proteins. However, curdlan doesn't trigger the hypersensitive response.
Curdlan belongs to the class of biological response modifiers that enhance or restore normal immune defenses, including antitumor, anti-infective, anti-inflammatory, and anticoagulant activities. CrdS is an integral inner membrane protein with seven transmembrane (TM) helices, one non-membrane-spanning amphipathic helix and a Nout–Cin disposition Sulfation of Curdlan. Acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV) is a severe disease that can destroy the body’s immune system, so the discovery of methods to prevent AIDS infection is of great importance. Our Solution
What is Curdlan?
ENZYMES + METABOLIC PATHWAY
Using Bacteria: E. coli
Using Yeast: S. cerevisiae
Effect on the plant
Now, why choose curdlan to replace copper sulfate?
Other useful properties of Curdlan