Difference between revisions of "Team:Bordeaux/Description"

Latest revision as of 02:24, 19 September 2015

IGEM Bordeaux 2015

Our Solution: Curdlan

What is Curdlan?

Glucan molecules are polysaccharides of D-glucose monomers linked by glycosidic bonds and one of them is called Curdlan , a (1→3)-β-D-glucan. This molecule is a linear homopolymer which may have as many as 12,000 glucose units. It is naturally produced by Agrobacterium sp. ATCC31749 which uses it as an Extracellular PolySaccharides (EPS) in it's capsule [1]. The capsule formation is correlated with cell aggregation (floc formation) and it is suggested that the capsule and floc formation together function as protective structures in cases of Nitrogen-starvation of the post-stationary phase. The protective effects for the bacteria are due to the fact that Curdlan forms a capsule that completely surrounds the outer cell surface of bacteria.

« Ok and how will Curdlan be useful to you? »

« Let me explain our purpose. »

Curdlan belongs to the class of biological response modifiers that enhance or restore normal natural defenses. For example, it can have antitumor, anti-infective, anti-inflammatory, and anticoagulant activities (see other properties of Curdlan). In particular, this (1→3)-β-glucan can stimulate plant natural defenses.

Figure 1: Representation of the Curdlan molecule showing the beta 1,3 links between the glucose units

More precisely, applied to grapevine plants, sulfated ß-glucans induce the accumulation of phytoalexins (organic antimicrobial substances) and the expression of a set of Pathogenesis-Related proteins . In plants, the fact that oligosaccharides must carry crucial sulfates for their biological function suggests that chemical sulfation of oligosaccharides can improve their biological properties. In recents studies, compared to Laminarin (ß-glucan), its sulfated derivative triggered an enhanced immunity against P. viticola in V. vinifera and a stronger immunity against TMV in Nicotiana tabacum. The results indicate that the chemical modification of an elicitor, such as sulfated derivative of ß-glucans, could improve its resistance-inducer efficiency. Moreover, if a ß-glucan is a substrate for plant (1→3)-β-glucanase, its sulfation clearly protects the molecule from its enzymatic degradation. Thus, a basal activity of plant glucanases can degrade ß-glucans and consequently releases short inactive ß-glucans; whereas sulfated derivatives still remain active molecules during a longer period. This might explain the higher resistance induced by ß-glucan sulfates compared to ß-glucans.

Furthermore, non-sulfated Curdlan doesn't trigger the response through a mutant gene: pmr4. This mutant is resistant to mildew infections but is unable to induce Pathogenesis-Related proteins expression . Also, activation of a Pathogenesis-Related protein called PR1 in grapevine is regulated by the salicylic acid signaling pathway . The lack of PR1 expression in non-sulfated Curdlan-treated grapevine could be explained by a negative feedback of glucan. This is demonstrated by the study of a double mutant of pmr4 which restore the susceptibility to mildew. It suggests that linear (1→3)-β-glucan negatively regulates the salicylic acid pathway. So, sulfation of the glucan would counteract the negative feedback effect. [16]

To conclude, activation of natural defenses before the invasion of pathogens is a way to improve the plant resistance against infection and to reduce the use of chemicals products.

« Ok I understand the value of producing Curdlan. How will you proceed? »

« That is a good question, let me tell you how we approached the subject! »

In Agrobacterium , three genes (crdA, crdS and crdC) are required for Curdlan production. The putative operon crdASC contains crdS, encoding (1→3)-β-glucan synthase catalytic subunit, flanked by two additional genes : crdA and crdC. The first assists translocation of the nascent polymer across the cytoplasmic membrane and the second assists the passage of the nascent polymer across the periplasm. Finally we would like to sulfate our Curdlan molecules chemically in order to enhance its effects on the activation of the plant natural defenses since it has been shown that sulfated curdlan is much more effective (see previous page [3]). However, all Curdlan biosynthesis is dependent of nitrogen starvation and various parameters. We want to simplify all of this.

Figure 2: Schematic representation of the (1→3)-β-glucan synthetic pathway [2]

Before starting the project, we took a few weeks to decide which host organism we would use and how they could be useful. To begin with, we looked at three different organisms: Escherichia coli , Bacillus subtilis and Saccharomyces cerevisiae and compared their β-glucan metabolic pathways. We rapidly eliminated Bacillus subtilis from our possible hosts due to its lack of enzymes involved in the metabolic pathway of (1→3)-β-glucans (Figure 5). However, we found that Saccharomyces cerevisiae naturally produces Curdlan in its cell wall, like Agrobacterium. Furthermore, Escherichia coli is only missing one enzyme (the β-Glucan synthase) to synthethize Curdlan. We therefore concluded that we could keep these two organisms: one where we would overexpress the (1→3)-β-glucan synthase using a constitutive promoter and one where we would insert the ability to create Curdlan by adding the enzyme that is needed throught the crdASC putative operon.

Literature Cited:

[1] M. McIntosh (2005) Curdlan and other bacterial (1→3)-β-D-glucans mini review. Appl Microbiol Biotechnol 68: 163–173

[2] M. McIntosh (2012) Recent advances in curdlan biosynthesis, biotechnological production, and applications. Appl Microbiol Biotechnol 93:525–531

[3] Adrien Gauthier (2014)The Sulfated Laminarin Triggers a Stress Transcriptome before Priming the SA- and ROS-Dependent Defenses during Grapevine’s Induced Resistance against Plasmopara viticola. Plos one 93:525–531

To sum it up, we would like to produce Curdlan in Escherichia coli or Saccharomyces cerevisiae and then sulfate it before spraying it on grapevine leaves as a preventive treatment against Downy mildew infections. That way vineyards can continue to produce good wine and make everyone happy.

Using Bacteria:
Escherichia coli

First, we decided to produce Curdlan with Escherichia coli , because Agrobacterium tumefasciens because both of them are Gram negative bacteria and have a lot of membrane similarity. Moreover Escherichia coli is a simple bacteria that can be grown and cultured easily and inexpensively in a laboratory unlike Agrobacterium . Indeed, Curdlan production in Agrobacterium requires Nitrogen Starvation and its growth parameters need to be relatively precise. This is not the case with E. coli in our project.

Since E. coli naturally produces UDP Glucose (metabolite number 3 on Figure 5), adding the (1→3)-β-glucan synthase would theoretically allow Curdlan production. We therefore inserted the three genes which code for the β-glucan synthase and metabolic transporters in Agrobacterium (crdASC) into E. coli . However, since our gene sequences for crdA, crdS, and crdC originally come from Agrobacterium we decided to optimize our gene codons for E. coli with IDT codon optimization tool in order to make sure that our gene would correctly be expressed. Furthermore, we decided to place the genes under an easier control than N-starvation by using a promoter which is activated in stationary phase. (osmY, BBa_J45992 characterized by MIT in 2006). This should theoretically allow maximum production in simple conditions.

All three genes were synthesized by IDT separately with the correct ends to allow integration in the iGEM plasmids and an easy creation of our biobricks. We planned on amplifying our fragments by PCR and creating different biobricks with different assemblies of our genes and plasmids in order to find the effect of each gene on Curdlan production and to verify the efficiency of our promoter.

In order to have control over the different culture perameters we also decided to use M63 culture medium. This is the medium which is used for Curdlan production in Agrobacterium and is also interesting since it is a minimal medium which allows us to easily vary parameters and optimize production. Furthermore, since Curdlan is a glucose derivative, being able to control the amount of sugar in the medium is interesting for production optimization. We plan on testing the effect of the following parameters on Curdlan production:

❃ Different mediums (minimal and complete)

❃ Different stationary phase temperature: by reducing the temperature we might be able to reduce growth and obtain more Curdlan

❃ Glucose shot during stationary phase at different times

❃ Lowering pH in stationary phase to reduce cell division

Using Yeast:
Saccharomyces cerevisiae

Yeast cell walls are naturally made up of various layers which are represented in the following diagram. First there is a layer of chitin, then a layer of β-glucans and finally a mixed layer of proteins and mannan. Commonly, the yeast cell wall is made of 5-10% of (1→6)-β-glucans and 50-55% of a mix of (1→3)-β-glucans and (1→3)-β-glucans.

Figure 3: Schematic representation of the yeast's cell wall

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 (1→6)-β-glucans(1→3)-β-D-glucans. (aimanianda et al 2009) In order to separate the two compounds, we plan on using (1→6)-β-glucanases in order to obtain a solution of (1→3)-β-glucans and therefore our Curdlan molecule.

We therefore decided to over-express the Curdlan metabolic pathway by inserting into yeast (INVSC1 strain ) an inducible promoter (Gal1) for the β-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.

To do this , we will extract the FKS1 gene from yeast DNA by amplifying the genomic DNA by PCR. We will then insert FKS1pYES2 plasmid with the Gal1 inductive promoter to then integrate the modified plasmid in Saccharomyces cerevisiae and boost the production of Curdlan. This strategy did not work. We then tried to put a inductive promoter ahead of the relevant gene by homologous recombination. We put the Gal1 promoter and a selective gene HIS3 (to select our successful transformants) in front of FKS1. Gal1 promoter and HIS3 was extract by PCR from pFA 6a-HIS3MX6-pGAL-3HA . On the other hand, we will integrate the FKS1 gene into the iGEM plasmid pSB1C3 to get our BioBrick that we'll send to Boston. This genetic construction with HIS3 gene and GAL1 promoter was inserted in pSB1C3 by Gibson assembly. However, site-directed mutagenesis may be necessary when integrating the gene into the plasmid because there are restriction sites (EcoR1) that are unwanted within the HIS3 gene.

« It's very clear now. It looks like a cool project. But, i have a last question: why did you choose this subject? »

« Because, as we explained on the problem page, Downy Mildew is a real problem for the Aquitaine region. This is why we wanted to find an ecological solution to this problem. And also, we are SWAG (Secretly We Adore Glucans) »

Other useful properties of Curdlan

Curdlan, the linear (1→3)-β-glucan from Agrobacterium, has unique rheo-logical and thermal gelling properties. It is neutral and insoluble in water and if it is heated in an aqueous suspension, it adopts simple helical conformations (55-80°C) or a triple helical connected conformation (80-130°C). [1] It then acts as a gelling agent and form two types of gels (low-set gel or high-set gel which have been documented by Zhang et al [3]). Apart from being tasteless, colourless and odourless, its advantages are that, in contrast to cold-set gels (e.g. gelatin, gellan, carrageenan) and heat-set gels (e.g. konjac glucomannan, methylcellulose), the heating process alone produces different forms of curdlan gels with different textural qualities, physical stabilities and water-holding capacities. Curdlan gels are widely used in the food industry as a food additive ( E424 ) and to develop new food products (e.g. freezable tofu noodles). It is also used in calorie-reduced food, since there are no digestive enzymes for Curdlan in the upper alimentary tract, and Curdlan can be used as a fat substitute [4]. The safety of Curdlan has been assessed in animal studies and in vitro tests [4,5] and it is approved for food use in Korea, Taiwan and Japan as an inert dietary fibre. It is registered in the United States as a food additive [7]

Usage as an essential ingredient:
Jellies	Gelling agent stable against heating and freezing-thawing	1-5%
Fabricated food Noodle-shaped tofu Processed tofu Thin-layered gel food Konjac-like gel food Heat-resistant cheese food	Gelling agent stable against heating and freezing-thawing	1-10%
Edible films	Film formation	30-100%
Dietetic foods	Low-energy ingredient

Curdlan has also found applications in non-food sectors. Its water-holding capacity is applied in the formulation of “superworkable” concrete, where its enhanced fluidity prevents cement and small stones from segregating [8]. It has also been proposed as an organic binding agent for ceramics [9]. In addition, curdlan gels have medical and pharmacological potential, for example in drug delivery through sustained and diffusion-controlled release of the active ingredient. [10]

Furthermore, Curdlan derivatives are members of a class of compounds known as biological response modifiers that enhance or restore normal natural defenses. Useful properties include antitumor, anti-infective, anti-inflammatory, and anticoagulant activities [11]. Hydrolysed Curdlan with a degree of polymerisation <50 are not effective anti-tumor agents but the carboxymethyl ether and the sulphate and phosphate esters of Curdlan, show an enhanced biological activity [12]. Furthermore, Curdlan sulphate has anti-HIV activity [13] and inhibitory effects on the development of malarial parasites in vitro [14]. All the other Curdlan clinical applications in cancer, diabetes, hypertension, hypertriglyceridemia etc. are listed here. Curdlan also has potential for exploitation as a new biomaterial based on the self-assembling ability of (1→3)-β-glucan-megalosaccharides (DP 30–45) to form single, hexagonal, lamellar nanocrystalline structures (∼8–9 nm thick) containing water of crystallization after heating to 90°C [15]. Manipulation of the conditions for self-assembly may allow the engineering of new materials.

However, more research is needed for the further development of these useful properties, in particular by reducing the cost of production. This may involve the use of cheaper C sources, optimization of fermentation conditions, development of higher Curdlan-yielding strains, or manipulation of Curdlan synthesis and/or regulatory genes. [1]

Literature Cited:

[1] M. McIntosh (2005) Curdlan and other bacterial (1→3)-β-D-glucans mini review. Appl Microbiol Biotechnol 68: 163–173

[2] R. Zhang and K. J. Edgar (2014) Properties, Chemistry, and Applications of the Bioactive Polysaccharide Curdlan. American Chemical Society

[3] Zhang HB, Nishinari K, Williams MAK, Foster TJ, Norton IT (2002) A molecular description of the gelation mechanism of curdlan. Int J Biol Macromol 30:7–16

[4] Nishinari K, Zhang H (2000) Curdlan. In: Phillips GO, Williams PA (eds) Handbook of hydrocolloids. CRC, Boca Raton, pp 269–286

[5] Spicer EJF, Goldenthal EI, Ikeda T (1999) A toxicological assessment of curdlan. Food Chem Toxicol 37:455–479

[6] (2000) WHO food additives series. In: WHO (ed) 53rd Meeting of the joint FAO/WHO expert committee on food additives. JEFCA/WHO, Geneva

[7] 21 CFR 172. Food additives permitted for direct addition to food for human consumption: curdlan. Federal Register 61:65941

[8] (1996) Bioproducts: bio-concrete. BioIndustry 13:56–57

[9] Harada T (1992) The story of research into curdlan and the bacteria producing it. Trends Glycosci Glycotechnol 4:309–317

[10] Kanke M, Tanabe E,(1995) Application of curdlan to controlled drug delivery. III. Drug release from sustained release suppositories in vitro. BiolPharm Bull 18:1154–1158

[11] Janeway CA, Medzhitov R (2002) Innate immune recognition. Annu Rev Immunol 20:197–216

[12] Toida T, Chaidedgumjorn A, Linhardt RJ (2003) Structure and bio- activity of sulphated polysaccharides. Trends Glycosci Glycotechnol 15:29–46

[13] Jagodzinski PP, Wiaderkiewicz R (1994) Mechanism of the inhibitory effect of curdlan sulphate on HIV-1 infection in vitro. Virology 202:735–745

[14] Evans SG, Morrison D, Kaneko Y, Havlik I (1998) The effect of curdlan sulphate on development in vitro of Plasmodium falciparum. Trans R Soc Trop Med Hyg 92:87–89

[15]Chanzy H, Vuong R (1985) Ultrastructure and morphology of crystalline polysaccharides. In Atkins EDT (ed) Polysaccharides: topics in structure and morphology. Macmillan, London, pp 41–71

[16]Menard R, Alban S, (2004) β-1,3 Glucan Sulfate, but not β-1,3 Glucan, Induces the Salicylic Acid Signaling Pathway in Tobacco and Arabidopsis

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+<!-- DESCRIPTION ---------------------------------------------------------------------------------------------- -->
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-            <h3>The Problem </h3>
-            <h6 align="justify"> This year, iGEM Bordeaux’s project is focused on Downy Mildew </h6>
+        <h3> Our Solution: Curdlan </h3>
-            <p align="justify" style="text-indent: 3vw;"> This disease, caused by an oomycete (fungus-like eukaryotic microorganism) called <b> <i> Plasmopara viticola </i> </b>,  is unfortunately famous in the Aquitaine region because it affects tens of hectares of Bordeaux vineyards every year and <b> threatens wine production </b> <b> (How much?) </b>. It was originally observed in the United States of America in 1834 and has been most abundantly found in the northern and midwestern areas of the United States. Shortly after, the pathogen was introduced in European countries where it played a devastating role in the yield and production of their wine. In <b>1878</b>, the <b>first cases of Downy mildew were observed in France </b> (in the region of Lyon) and also in Swizerland and Italy. Common symptoms include necrosis of the stem or shoot, discoloration, brown spotting and yellowish-green tips of the leaves and mycelium invasion of the grapes. While some North American species have become resistant to this parasite, European species such as <i> Vitis vinifera </i>  (the grapevine used for wine) are extremely sensitive. Depending on the year, production of grapes in France has been estimated to be at a loss of 50% or more <b> ref </b> and the Aquitaine region is particularly affected due to the favorable climate and the economic importance of the wine industry. Thus, Downy mildew has been considered the most <b> devastating disease </b> caused by a <b> filamentous pathogen </b> to affect European vineyards and this has lead vineyards to search for effective measures to protect their vines. Unfortunately, most of these mesures have a bad environmental impact and pollute the surrounding soils.
+        <h5 align="left"> What is Curdlan? </h5>
-</p>
-            <br>
-            <h6 align="justify"> In 2015, vineyards are still threatened by the disease </h6>
-            <p align="justify" style="text-indent: 3vw;"> Our iGEM team has been following this year's effect of mildew closely reading the official vineyard mildew bulletins available on the vinopole website. It is clearly shown that there is a significant increase of mildew infection on parcels that haven't been treated with copper sulfate compared to those that have been treated. Furthermore, the infection of mildew on treated parcels appears to be  much more easily controled on parcels treated with copper sulfate. Evidently, without any alternative treatment, wine production in the region would be affected and this shows just how important our project is! </p>
-        <p align="justify"> Different models (Caffi model, Potential systems model) take into account pluviometry, temperature, relative humidity and plant morphology to decide when are the best moments to apply the fungicides. However, even if these models have allowed vinyards to drastically reduce the quantities of fungicides used, they still cause environmental and sanitary problems in the surrounding regions. </p>
-       </div>
-<!-- ------------------------------------------------------------------------------------------ -->
+        <p align="justify" style="text-indent: 3vw;"> Glucan molecules are <b>polysaccharides of D-glucose monomers linked by glycosidic bonds</b> and one of them is called <b>Curdlan </b>, a (1→3)-β-D-glucan. This molecule is a linear homopolymer which may have as many as 12,000 glucose units. It is <b>naturally produced by <i> Agrobacterium</b> sp. ATCC31749</i> which uses it as an Extracellular PolySaccharides (EPS) in it's capsule [1]. The capsule formation is correlated with cell aggregation (floc formation) and it is suggested that the capsule and floc formation together function as protective structures in cases of Nitrogen-starvation of the post-stationary phase. The protective effects for the bacteria are due to the fact that Curdlan forms a capsule that completely surrounds the outer cell surface of bacteria.</p>
-            <div class="col-lg-10 col-lg-offset-1">
-                  <div class="col-lg-8">
-<br> <br>
-                  <p align="justify" style="text-indent: 3vw;"> In the past few months (June 2015) there has been another violent attack of mildew on the grapevines in the Aquitaine region. Up to 60% of wine grapes have been infected on certain parcels and the vice president of the agriculture chamber, Patrick Vasseur, hasn't been underestimating the economic significance this could have since the wine production will evidently be affected. He calls the situation <i>"exceptional" </i> since <i> "even the main branches are affected"</i> </p>
-                  <p align="justify"> Serge Audubert, head of 3 castles in the region and owning a total of 24 ha, has been watching the effects on his land. On his 17 ha of château-laborde grapevines, in Saint-Médard-de-Guizières, 2ha are severly touched.<i> « the leaves, the branches, the grapes, everything is affected. We are going to loose at least 50% of the grapes on these 2 ha. » </i> On the first of may, this vineyard observed a spot on a branch, nothing severe especially since the « Bulletin de santé du végétal » (plant health review) which came out a few days before clearly states that the conditions aren't favorable for contaminations. As a precaution, Serge Audubert starts his preventive treatments on the 7th of may. On the 15th of May, the outburst starts, shocking the entire region: <i> « I have been living here since 1987. I have never seen something like this. Informatics models were supposed to alert us when mildew evolution becomes dangerous. » </i> </p>
-                  <br>
-                  </div>
-                  <div class="col-lg-2">
+            <div class="col-lg-6"> <br>
-                  <br>
+            <h6 align="justify"> <i> <FONT color="#8b008b"> « Ok and how will Curdlan be useful to you? »  </FONT> </i> </h6>
-                  <img style="width:30vw;height:35vw" src="https://static.igem.org/mediawiki/2015/thumb/7/75/Bordeaux_parcels.png/484px-Bordeaux_parcels.png">
+            <h6 align="justify"> <i> <FONT color="#00843c"> « Let me explain our purpose. » </FONT> </i> </h6> <br>
-                  </div>
+            <p align="justify" style="text-indent: 3vw;"> Curdlan belongs to the class of biological response modifiers that <b>enhance or restore normal natural defenses</b>. For example, it can have antitumor, anti-infective, anti-inflammatory, and anticoagulant activities (see other properties of Curdlan). In particular, this (1→3)-β-glucan can <b>stimulate plant natural defenses</b>. </p>
-             </div>
+            </div>
-<!-- ------------------------------------------------------------------------------------------ -->
+            <div class="col-lg-6">
+            <img style= "width:46vw; height:15vw;" src="https://static.igem.org/mediawiki/2015/thumb/9/9f/Bordeaux_Curdlan.png/800px-Bordeaux_Curdlan.png">
+<p class="reference" align ="justify"> <b> Figure 1: Representation of the Curdlan molecule showing the beta 1,3 links between the glucose units</b> </p>
+               </div>
+        </div>
-         <div class="col-lg-10 col-lg-offset-1">
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-                  <br> <br> <br> <br>
-                  <img style= "width:35vw; height:30vw;"   src='https://static.igem.org/mediawiki/2015/thumb/c/c8/Bordeaux_mildew_infection.png/727px-Bordeaux_mildew_infection.png' >
-                  </div>
-                  <div class="col-lg-7">
+<!-- PHYTOALEXINS ---------------------------------------------------------------------------------------------- -->
-                  <br> <br>
-                  <h6 align="justify"> Infection Mode of Downy Mildew</h6>
+<div class="col-lg-10 col-lg-offset-1">
-                  <p align="justify" style="text-indent: 3vw;"> Downy mildew requires optimum conditions to reproduce and infect as warm, moist, and humid environment. </p>
+<p align="justify" style="text-indent: 3vw;"> More precisely, applied to grapevine plants, <b> sulfated ß-glucans </b> induce the <b> accumulation of phytoalexins </b> (organic antimicrobial substances) and the <b> expression of a set of Pathogenesis-Related proteins </b>. In plants, the fact that oligosaccharides must carry crucial sulfates for their biological function suggests that <b>chemical sulfation</b> of oligosaccharides <b>can improve their biological properties.</b> In recents studies, compared to Laminarin (ß-glucan), its sulfated derivative triggered an enhanced immunity against <i>P. viticola</i> in <i>V. vinifera</i> and a stronger immunity against TMV in <i>Nicotiana tabacum</i>. The results indicate that the chemical modification of an elicitor, such as sulfated derivative of ß-glucans, could improve its resistance-inducer efficiency. Moreover, if a ß-glucan is a substrate for plant (1→3)-β-glucanase, its <b>sulfation</b> clearly <b>protects the molecule</b> from its enzymatic degradation. Thus, a basal activity of plant glucanases can degrade ß-glucans and consequently releases short inactive ß-glucans; whereas sulfated derivatives still remain active molecules during a longer period. This might explain the higher resistance induced by ß-glucan sulfates compared to ß-glucans. </p>
-<p align="justify"> In winter, <i> Plasmopara viticola </i> is present on dead leaves on the ground as oospores. They are inactive and do not produce any symptoms. When rain falls during spring, these eggs grow and release zoospores when the temperature exceeds 11 degrees. The zoospores will be able to spread and infect the plant's upper tissues through rainwater's splashes. </p>
-                  <p align="justify">The primary contamination begins by the emission of a filament through the stomatal area where the parasite begins to develop sinkers from which is formed the mycelial network. These sinkers help to feed <i>Plasmopara viticola </i> by stealing the plant's nutrients, which creates discolored and yellowish areas on the it's leaves called “oil stains”. After, on the bottom, conidiophores and conidia are formed. These symptoms cause damages to the leaves’ tissues and affect the plant’s photosynthetic ability, which slows down the maturity of the plant.</p>
+        <p align="justify"style="text-indent: 3vw;">  Furthermore, non-sulfated Curdlan doesn't trigger the response through a mutant gene: pmr4. This <b>mutant</b> is resistant to mildew infections but is <b> unable to induce Pathogenesis-Related proteins expression </b>. Also, activation of a Pathogenesis-Related protein called PR1 in grapevine is regulated by the <b> salicylic acid signaling pathway </b>. The lack of PR1 expression in non-sulfated Curdlan-treated grapevine could be explained by a negative feedback of glucan. This is demonstrated by the study of a double mutant of pmr4 which restore the susceptibility to mildew. It suggests that linear (1→3)-β-glucan negatively regulates the salicylic acid pathway. So, <b>sulfation of the glucan would counteract the negative feedback effect. </b> [16]</p>
-         <p align="justify"> During the secondary contamination, the conidia are transformed into zoospores that contaminate the surrounding tissues, weakening the plant even more and creating unreparable lesions. </p>
-                 </div>
+         <p align="justify" style="text-indent: 3vw;">  To conclude, activation of natural defenses before the invasion of pathogens is a way to improve the plant resistance against infection and to reduce the use of chemicals products.  </p>
+<br>
+        <h6 align="justify"> <i> <FONT color="#8b008b"> « Ok I understand the value of producing Curdlan. How will you proceed? » </FONT> </i> </h6>
+        <h6 align="justify"> <i> <FONT color="#00843c"> « That is a good question, let me tell you how we approached the subject! » </FONT> </i> </h6>
           </div>
+<!-- GENES ---------------------------------------------------------------------------------------------- -->
          <div class="col-lg-10 col-lg-offset-1">
+                <div class="col-lg-6">   <br> <br> <br>
+                <p align="justify" style="text-indent: 3vw;"> In <i> Agrobacterium </i>, <b>three genes (crdA, crdS and crdC) are required for Curdlan production</b>. The putative operon crdASC contains <b>crdS, encoding (1→3)-β-glucan synthase catalytic subunit</b>, flanked by two additional genes : crdA and crdC. The first assists translocation of the nascent polymer across the cytoplasmic membrane and the second assists the passage of the nascent polymer across the periplasm. Finally we would like to <b>sulfate</b> our Curdlan molecules chemically in order to <b>enhance its effects on the activation of the plant natural defenses</b> since it has been shown that sulfated curdlan is much more effective (see previous page [3]). <b>However</b>, all <b>Curdlan biosynthesis is dependent of nitrogen starvation</b> and various parameters. We want to <b>simplify</b> all of this. </p>
+                </div>
+                <div class="col-lg-6"> <br> <br>
+<img style= "width:40vw; height:15vw;"src="https://static.igem.org/mediawiki/2015/a/ad/Bordeaux_biosyntheseV2.png">
+<p class="reference" align ="center"> <b> Figure 2: Schematic representation of the (1→3)-β-glucan synthetic pathway [2] </b> </p>
+                </div>
+        </div>
+<!-- PROJECT ---------------------------------------------------------------------------------------------- -->
+        <div class="col-lg-10 col-lg-offset-1"> <br> <br>
+        <p align="justify" style="text-indent: 3vw;">  Before starting the project, we took a few weeks to decide <b>which host organism we would use</b> and how they could be useful. To begin with, we looked at three different organisms: <i> Escherichia coli </i>, <i> Bacillus subtilis </i> and <i> Saccharomyces cerevisiae </i> and compared their β-glucan metabolic pathways. We rapidly eliminated <i> Bacillus subtilis </i> from our possible hosts due to its lack of enzymes involved in the metabolic pathway of (1→3)-β-glucans (Figure 5). However, we found that <b><i>Saccharomyces cerevisiae</i> naturally produces Curdlan</b> in its cell wall, like <i>Agrobacterium</i>. Furthermore, <b><i> Escherichia coli </i> is only missing one enzyme</b> (the <b>β-Glucan synthase</b>) to synthethize Curdlan. We therefore concluded that we could keep these two organisms: one where we would overexpress the (1→3)-β-glucan synthase using a constitutive promoter and one where we would insert the ability to create Curdlan by adding the enzyme that is needed throught the crdASC putative operon.</p>
+        <p class="reference" align="left"> <b>Literature Cited: </b> </p>
+        <p class="reference" align="left"> [1] M. McIntosh (2005) Curdlan and other bacterial (1→3)-β-D-glucans mini review. <I> Appl Microbiol Biotechnol 68: 163–173 </i> </p>
+        <p class="reference" align="left"> [2] M. McIntosh (2012) Recent advances in curdlan biosynthesis, biotechnological production, and applications. <i> Appl Microbiol Biotechnol 93:525–531 </i></p>
+        <p class="reference" align="left"> [3] Adrien Gauthier (2014)The Sulfated Laminarin Triggers a Stress Transcriptome before Priming the SA- and ROS-Dependent Defenses during Grapevine’s Induced Resistance against Plasmopara viticola. <i>Plos one 93:525–531</i></p>
+        <br> <br> <br>
+        <p style= "font-size:150%"> <FONT color="#00843c"> To sum it up, we would like to <b>produce Curdlan</b> in <b><i> Escherichia coli </i> </b>or <b><i> Saccharomyces cerevisiae </i></b> and then <b>sulfate</b> it before spraying it on grapevine leaves as a <b>preventive treatment</b> against Downy mildew infections. That way vineyards can continue to produce <b>good wine</b> and <b>make everyone happy</b>. </FONT></b> </p>
+        <br> <br>
+        </div>
+   </div>
+</section>
+<!-- ------------------PHOTO--------------------------------------------------------------------------------- -->
+<section id="statistic" class="parallax" style="background-image: url(https://static.igem.org/mediawiki/2015/thumb/d/d4/Bordeaux_Photos1_boites.001.png/800px-Bordeaux_Photos1_boites.001.png)";>
+        <p align="justify"> </p>
+</section>
+<!-- ------------------E COLI--------------------------------------------------------------------------------- -->
+<section align="center">
+    <div class="content-wrapper">
+        <div class="col-lg-10 col-lg-offset-1">
+                <div class="col-lg-6">
+                <h5> Using Bacteria: <br> <i> Escherichia coli </i> </h5>
+                <p align="justify" style="text-indent: 3vw;"> First, we decided to produce Curdlan with <b><i> Escherichia coli </i></b>, because <i> Agrobacterium tumefasciens</i> because both of them are Gram negative bacteria and have a lot of <b>membrane similarity</b>. Moreover <i> Escherichia coli </i> is a simple bacteria that can be <b>grown and cultured easily and inexpensively</b> in a laboratory unlike <i> Agrobacterium </i>. Indeed, Curdlan production in <i> Agrobacterium </i> requires Nitrogen Starvation and its growth parameters need to be relatively precise. This is not the case with <i> E. coli </i> in our project. </p>
+                <p align="justify" style="text-indent: 3vw;"> Since <i> E. coli </i> naturally produces UDP Glucose (metabolite number 3 on Figure 5), adding the (1→3)-β-glucan synthase would theoretically allow Curdlan production. We therefore inserted the three genes which code for the β-glucan synthase and metabolic transporters in <i> Agrobacterium </i> (crdASC) into <i> E. coli </i>. However, since our gene sequences for crdA, crdS, and crdC originally come from <i> Agrobacterium </i> we decided to <b>optimize our gene codons</b> for <i> E. coli </i> with IDT codon optimization tool in order to make sure that our gene would correctly be expressed. Furthermore, we decided to place the genes under an easier control than N-starvation by using a <b>promoter</b> which is <b>activated in stationary phase</b>. (osmY, <a href= "http://parts.igem.org/Part:BBa_J45992" style=" color: #8b008b;"> BBa_J45992  </a> characterized by MIT in 2006). This should theoretically allow maximum production in simple conditions. </p>
+<img style= "width:30vw; height:8vw; align:center;" src= "https://static.igem.org/mediawiki/2015/7/79/Bordeaux_Biobrick.jpg">
+                <p align="justify" style="text-indent: 3vw;"> All three genes were synthesized by IDT separately with the correct ends to allow integration in the iGEM plasmids and an easy creation of our biobricks. We planned on amplifying our fragments by PCR and creating different biobricks with different assemblies of our genes and plasmids in order to find the <b>effect of each gene on Curdlan production</b> and to <b>verify the efficiency of our promoter</b>. </p>
+                <p align="justify" style="text-indent: 3vw;"> In order to have control over the different culture perameters we also decided to use M63 culture medium. This is the medium which is used for Curdlan production in <i> Agrobacterium </i> and is also interesting since it is a minimal medium which allows us to easily vary parameters and optimize production. Furthermore, since Curdlan is a glucose derivative, being able to control the amount of sugar in the medium is interesting for production optimization. <b>We plan on testing the effect of the following parameters on Curdlan production</b>: </p>
+<p align="justify"> &#10051;  Different mediums (minimal and complete)</p>
+<p align="justify"> &#10051;  Different stationary phase temperature: by reducing the temperature we might be able to reduce growth and obtain more Curdlan </p>
+<p align="justify"> &#10051;  Glucose shot during stationary phase at different times </p>
+<p align="justify"> &#10051;  Lowering pH in stationary phase to reduce cell division </p>
+                </div>
+<!-- --------------S. CEREVISIAE----------------------------------------------------------------- -->
+                <div class="col-lg-6 ol-lg-offset-1">
+                <h5> Using Yeast: <br> <i> Saccharomyces cerevisiae </i> </h5>
+                <p align="justify" style="text-indent: 3vw;">  Yeast cell walls are naturally made up of various layers which are represented in the following diagram. First there is a layer of chitin, then a layer of β-glucans and finally a mixed layer of proteins and mannan. Commonly, the yeast cell wall is made of 5-10% of (1→6)-β-glucans and 50-55% of a <b>mix of (1→3)-β-glucans and (1→3)-β-glucans.</b> </p>
+                <img style= "width:38vw; height:15vw;"  src="https://static.igem.org/mediawiki/2015/thumb/f/f8/Bordeaux_yeast_wall.png/800px-Bordeaux_yeast_wall.png" >
+                <p class="reference" align ="justify"> <b> Figure 3: Schematic representation of the yeast's cell wall </b> </p>
+                <p align="justify" style="text-indent: 3vw;">   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 (1→6)-β-glucans(1→3)-β-D-glucans. (aimanianda et al 2009) In order to separate the two compounds,  we plan on using <b>(1→6)-β-glucanases</b> in order to obtain a solution of (1→3)-β-glucans and therefore our Curdlan molecule. </p>
+                <p align="justify" style="text-indent: 3vw;"> We therefore decided to over-express the Curdlan metabolic pathway by inserting into yeast (<i>INVSC1</i> strain ) an inducible promoter (<i>Gal1</i>) for the β-glucan synthase gene (<i>Fks1</i>)  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. </p>
+                <p align="justify" style="text-indent: 3vw;"> To do this , we will extract the <b><i>FKS1</i> gene</b> from yeast DNA by amplifying the genomic DNA by PCR. We will then insert <i>FKS1</i gene in one hand, into <b>pYES2 plasmid</b> with the <i>Gal1</i> inductive promoter to then integrate the modified plasmid in <i>Saccharomyces cerevisiae </i> and boost the production of Curdlan. This strategy did not work. We then tried to put a inductive promoter ahead of the relevant gene by homologous recombination. We put the <i>Gal1</i> promoter and a selective gene <i>HIS3</i> (to select our successful transformants) in front of <i>FKS1</i>.  <i>Gal1</i> promoter and <i>HIS3</i> was extract by PCR from <b>pFA 6a-HIS3MX6-pGAL-3HA</b> .
+On the other hand, we will integrate the <i>FKS1</i> gene into the iGEM plasmid pSB1C3 to get our BioBrick that we'll send to Boston. This genetic construction with <i>HIS3</i> gene  and <i>GAL1</i> promoter was inserted in pSB1C3 by Gibson assembly. However, site-directed mutagenesis may be necessary when integrating the gene into the plasmid because there are restriction sites (EcoR1) that are unwanted within the <i>HIS3</i> gene. </p>
+                </div>
+        </div>
+<!-- --------------------------- END ---------------------------------------------------------------------- -->
+        <div class="col-lg-10 col-lg-offset-1">
+        <br>
 <br>
-        <p align="justify" style="text-indent: 3vw;"> Since repairing damaged tissues infected by downy mildew is impossible, the main solutions available to vinyards are preventive solutions, mainly through preventing primary infections. This is mainly done by spraying fungicides on the organs that are most infected: leaves and stems. The most efficient preventive treatment was discovered at the end of the 19th century: a solution made of copper sulfate also known as "Bouillie Bordelaise", the only treatment used until the end of the 20th century. Recently, synthetic fungicides have replaced this chemical treatment and more and more research is being done on alternative eco-friendly preventive treatments. </p>
-<br>
-<h6 align="justify"> Natural defenses of plants  </h6>
-<p align="justify" style="text-indent: 3vw;"> Immunization of plants is based on the same principle than humans: activate the natural defenses before contamination by the infectious agent. The concept is simple; it is to put the plant in contact with a molecule able to activate plant's natural defenses: <b> an elicitor </b> . In nature there are many elicitors produced by micro-organisms (exogenous elicitors) or by the plant itself when it is attacked (endogenous elicitors). The presence of an elicitor in the plant triggers a series of cellular reactions including the <b> production of molecules to strengthen the resistance of walls </b>, but also the <b> production of plant antibiotics </b> such as phytoalexins or defense proteins. These compounds have antifungal and antibacterial properties.
-The external application of a natural elicitor or a similar synthetic molecule thus results in the production of phytoalexins or defense proteins in the absence of any pathogen. The plant "immunized" is ready to fight back if attacked.
-First, the cell wall forms a physical barrier which prevents the penetration of most microbes. Then, if some pathogens are able to cross this wall, the infection depends on the ability of the plant to perceive and to trigger defense reactions that would prevent the development of the disease. This recognition is done with certain compounds, known as elicitors from the pathogen or plant. <b> The fixing of an elicitor to a plant cell receptor initiates a cascade of events that leads to the synthesis of defense compounds </b>. The best known are antimicrobial compounds such as <b> phytoalexins </b> and <b> Pathogenesis Related  proteins </b>. </p>
-<p align="justify"> There are two types of defenses:  "passive" and "active".
-Natural defense reactions of plants are passive when these components were preformed prior to infection. They remain active until the pathogen penetration.
-Active defenses are induced upon recognition of the pathogen and remain active during infection.</p>
-<br>
- <img style="width:50vw;height:30vw"
-src="https://static.igem.org/mediawiki/2015/thumb/a/ab/Bordeaux_MildewVSbeta1%2C3glucan.png/800px-Bordeaux_MildewVSbeta1%2C3glucan.png">
-<br>
-<br>
-<h6 align="justify" > Consequences of the infection </h6>
-<p align="justify" style="text-indent: 3vw;"> Delta-viniferin is a <b> grapevine phytoalexin </b> produced during infection by <i> Plasmopara viticola </i>.
-Phytoalexins are antimicrobial and often antioxidative substances synthesized by plants that accumulate rapidly at areas of pathogen infection. Phytoalexins produced in plants act as toxins to the organism. They damage the cell wall, delay maturation, disrupt metabolism or prevent reproduction of the pathogen. </p>
-<p align="justify"> When phytoalexin biosynthesis is inhibited, the susceptibility of infection in plant tissue increases showing its <b> importance in defense mechanism </b>. While, when a plant cell recognizes particles from damaged cells or from the pathogen, the plant launches a two-pronged resistance: a general short-term response and a delayed long-term specific response. </p>
-<p align="justify"> During the <b> short-term response </b>, the plant deploys reactive oxygen species such as superoxide and hydrogen peroxide to neutralize invading cells.
-The short-term response corresponds to the hypersensitive response, in which cells surrounding the site of infection are signaled to undergo <b> apoptosis </b> (programmed cell death), in order to prevent the spread of the pathogen to the rest of the plant. </p>
-<p align="justify"> The <b> long-term response </b>, also called systemic acquired resistance (SAR), permits a <b> communication </b> of damaged tissues with the rest of the plant using plant <b> hormones </b> such as jasmonic acid, ethylene, abscisic acid or salicylic acid. The reception of the <b> signal </b> leads to changes within the plant <b> inducing genes </b> that protect from more pathogen intrusion like genes coding enzymes involved in the production of phytoalexins. Also, if jasmonates or ethylene is released from the wounded tissue, neighboring plants also synthesize phytoalexins in response. </p>
-         </div>
+        <h6 align="justify"> <i> <FONT color="#8b008b">  « It's very clear now. It looks like a cool project. But, i have a last question: why did you choose this subject? » </FONT> </i> </h6>
+        <br>
+        <h6 align="justify"> <i> <FONT color="#00843c">  « Because, as we explained on the problem page, Downy Mildew is a real problem for the Aquitaine region. This is why we wanted to find an ecological solution to this problem. And also, we are SWAG (Secretly We Adore Glucans) »  </FONT> </i> </h6>
+        <br>
+        </div>
+   </div>
+</section>
+<!-- --------------------------- OTHER PROPERTIES --------------------------------------------------------- -->
+<section align="center">
+    <div class="content-wrapper">
+        <div class="col-lg-10 col-lg-offset-1">
+        <h5> Other useful properties of Curdlan </h5>
+        <div class="col-lg-7">
+        <p align="justify" style="text-indent: 3vw;"> Curdlan, the linear (1→3)-β-glucan from Agrobacterium, has unique <b>rheo-logical and thermal gelling properties</b>. It is neutral and insoluble in water and if it is heated in an aqueous suspension, it adopts simple helical conformations (55-80°C) or a triple helical connected conformation (80-130°C). [1] It then acts as a <b>gelling agent</b> and form two types of gels (low-set gel or high-set gel which have been documented by Zhang et al [3]). Apart from being tasteless, colourless and odourless, its advantages are that, in contrast to cold-set gels (e.g. gelatin, gellan, carrageenan) and heat-set gels (e.g. konjac glucomannan, methylcellulose), the heating process alone produces different forms of curdlan gels with different textural qualities, physical stabilities and water-holding capacities. Curdlan gels are widely used in the food industry as a food additive ( E424 ) and to develop new food products (e.g. freezable tofu noodles). It is also used in calorie-reduced food, since there are no digestive enzymes for Curdlan in the upper alimentary tract, and Curdlan can be used as a fat substitute [4]. The safety of Curdlan has been assessed in animal studies and in vitro tests [4,5] and it is approved for food use in Korea, Taiwan and Japan as an inert dietary fibre. It is registered in the United States as a <b>food additive</b> [7] </p>
+       </div>
+       <div class="col-lg-5"> <br> <br>
+<style type="text/css">
+.tg  {border-collapse:collapse;border-spacing:0;border-color:#ccc;border:none;}
+.tg td{font-family:Arial, sans-serif;font-size:14px;padding:10px 14px;border-style:solid;border-width:0px;overflow:hidden;word-break:normal;border-color:#ccc;color:#333;background-color:#fff;}
+.tg th{font-family:Arial, sans-serif;font-size:14px;font-weight:normal;padding:10px 14px;border-style:solid;border-width:0px;overflow:hidden;word-break:normal;border-color:#ccc;color:#333;background-color:#f0f0f0;}
+.tg .tg-rqa8{font-weight:bold;font-size:11px;font-family:Arial, Helvetica, sans-serif !important;;background-color:#f0f0f0;text-align:center}
+.tg .tg-p0g9{font-weight:bold;text-decoration:underline;font-size:11px;font-family:Arial, Helvetica, sans-serif !important;;text-align:center}
+.tg .tg-vuwi{font-size:11px;font-family:Arial, Helvetica, sans-serif !important;;text-align:center}
+</style>
+<table class="tg">
+  <tr>
+    <th class="tg-p0g9" colspan="3">Usage as an essential ingredient:</th>
+  </tr>
+  <tr>
+    <td class="tg-rqa8">Jellies</td>
+    <td class="tg-vuwi">Gelling agent<br>  stable against heating<br>  and freezing-thawing</td>
+    <td class="tg-vuwi">1-5%</td>
+  </tr>
+  <tr>
+    <td class="tg-rqa8">Fabricated food<br>  Noodle-shaped tofu<br>  Processed tofu<br>  Thin-layered gel food<br>  Konjac-like gel food<br>  Heat-resistant cheese food<br></td>
+    <td class="tg-vuwi">Gelling agent<br>  stable against heating<br>  and freezing-thawing</td>
+    <td class="tg-vuwi">1-10%</td>
+  </tr>
+  <tr>
+    <td class="tg-rqa8">Edible films</td>
+    <td class="tg-vuwi">Film formation</td>
+    <td class="tg-vuwi">30-100%</td>
+  </tr>
+  <tr>
+    <td class="tg-rqa8">Dietetic foods</td>
+    <td class="tg-vuwi">Low-energy ingredient </td>
+    <td class="tg-vuwi"></td>
+  </tr>
+</table>
+       </div>
+     </div>
+     <div class="col-lg-10 col-lg-offset-1">
+        <p align="justify" style="text-indent: 3vw;"> Curdlan has also found applications in non-food sectors. Its water-holding capacity is applied in the formulation of “superworkable” concrete, where its enhanced fluidity prevents cement and small stones from segregating [8]. It has also been proposed as an organic binding agent for ceramics [9]. In addition, curdlan gels have <b>medical and pharmacological potential</b>, for example in drug delivery through sustained and diffusion-controlled release of the active ingredient. [10]</p>
+        <p align="justify" style="text-indent: 3vw;"> Furthermore, Curdlan derivatives are members of a class of compounds known as biological response modifiers that enhance or restore normal natural defenses. Useful properties include <b>antitumor, anti-infective, anti-inflammatory, and anticoagulant activities</b> [11]. Hydrolysed Curdlan with a degree of polymerisation <50 are not effective anti-tumor agents but the carboxymethyl ether and the sulphate and phosphate esters of Curdlan, show an enhanced biological activity [12]. Furthermore, Curdlan sulphate has <b>anti-HIV activity</b> [13] and inhibitory effects on the development of malarial parasites in vitro [14]. All the other Curdlan clinical applications in cancer, diabetes, hypertension, hypertriglyceridemia etc. are listed <a href ="https://static.igem.org/mediawiki/2015/f/fb/Bordeaux_Clinical_Applications.pdf"> here</a>. Curdlan also has potential for exploitation as a new biomaterial based on the self-assembling ability of (1→3)-β-glucan-megalosaccharides (DP 30–45) to form single, hexagonal, lamellar nanocrystalline structures (∼8–9 nm thick) containing water of crystallization after heating to 90°C [15]. Manipulation of the conditions for self-assembly may allow the engineering of new materials. </p>
+        <p align="justify" style="text-indent: 3vw;"> However, more research is needed for the further development of these useful properties, in particular by reducing the cost of production. This may involve the use of cheaper C sources, optimization of fermentation conditions, development of higher Curdlan-yielding strains, or manipulation of Curdlan synthesis and/or regulatory genes. [1] </p>
+<!-- --------------------------------------------- BIBLIO OTHER  ---------------------------------------------------------- -->
+     <p class="reference" align="left"> <b>Literature Cited: </b> </p>
+<p class="reference" align="left"> [1] M. McIntosh (2005) Curdlan and other bacterial (1→3)-β-D-glucans mini review. <i>Appl Microbiol Biotechnol 68: 163–173</i> </p>
+<p class="reference" align="left"> [2] R. Zhang and K. J. Edgar (2014) Properties, Chemistry, and Applications of the Bioactive Polysaccharide Curdlan. <i>American Chemical Society</i>
+<p class="reference" align="left"> [3] Zhang HB, Nishinari K, Williams MAK, Foster TJ, Norton IT (2002) A molecular description of the gelation mechanism of curdlan. <i>Int J Biol Macromol 30:7–16</i></p>
+<p class="reference" align="left"> [4] Nishinari K, Zhang H (2000) Curdlan. In: Phillips GO, Williams PA (eds) Handbook of hydrocolloids. <i>CRC, Boca Raton, pp 269–286</i></p>
+<p class="reference" align="left"> [5] Spicer EJF, Goldenthal EI, Ikeda T (1999) A toxicological assessment of curdlan. <i>Food Chem Toxicol 37:455–479</i></p>
+<p class="reference" align="left"> [6] (2000) WHO food additives series. In: WHO (ed) 53rd Meeting of the joint FAO/WHO expert committee on food additives. <i>JEFCA/WHO, Geneva</i></p>
+<p class="reference" align="left"> [7] 21 CFR 172. Food additives permitted for direct addition to food for human consumption: curdlan.<i> Federal Register 61:65941 </i></p>
+<p class="reference" align="left"> [8] (1996) Bioproducts: bio-concrete. <i>BioIndustry 13:56–57</i></p>
+<p class="reference" align="left"> [9] Harada T (1992) The story of research into curdlan and the bacteria producing it. <i>Trends Glycosci Glycotechnol 4:309–317</i></p>
+<p class="reference" align="left"> [10] Kanke M, Tanabe E,(1995) Application of curdlan to controlled drug delivery. III. Drug release from sustained release suppositories in vitro.<i> BiolPharm Bull 18:1154–1158</i></p>
+<p class="reference" align="left"> [11] Janeway CA, Medzhitov R (2002) Innate immune recognition. <i>Annu Rev Immunol 20:197–216</i></p>
+<p class="reference" align="left"> [12] Toida T, Chaidedgumjorn A, Linhardt RJ (2003) Structure and bio-
+activity of sulphated polysaccharides. <i>Trends Glycosci Glycotechnol 15:29–46</i> </p>
+<p class="reference" align="left"> [13] Jagodzinski PP, Wiaderkiewicz R (1994) Mechanism of the inhibitory effect of curdlan sulphate on HIV-1 infection in vitro. <i>Virology 202:735–745 </i></p>
+<p class="reference" align="left"> [14] Evans SG, Morrison D, Kaneko Y, Havlik I (1998) The effect of curdlan sulphate on development in vitro of Plasmodium falciparum. <i>Trans R Soc Trop Med Hyg 92:87–89 </i></p>
+<p class="reference" align="left"> [15]Chanzy H, Vuong R (1985) Ultrastructure and morphology of crystalline polysaccharides. In Atkins EDT (ed) Polysaccharides: topics in structure and morphology. <i>Macmillan, London, pp 41–71</i> </p>
+<p class="reference" align="left"> [16]Menard R, Alban S, (2004) β-1,3 Glucan Sulfate, but not β-1,3 Glucan, Induces the Salicylic Acid Signaling Pathway in Tobacco and Arabidopsis </p>
+        </div>
+        <div class="col-lg-10 col-lg-offset-1">
+<br> <br>
+<h6> <a href= "https://2015.igem.org/Team:Bordeaux/Problem" style=" color: #FF5E00;"> Problem &#9754;  </a>   Previous Page . Next Page  <a href= "https://2015.igem.org/Team:Bordeaux/AchievementsNotebook" style=" color: #FF5E00;"> &#9755; Notebook & Protocols </h6>
+        </div> </div>
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