Difference between revisions of "Team:ShanghaiTechChina B/Proof"

 
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                         <h1>NO gradient in IBD patients, and the SNP releasing NO</h1>
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                         <div class="h1"">We have achieved following experiment results as our PROOF OF CONCEPT.</div>
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                         <h3 id="Introduction">Introduction</h3>
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                         <div class="p">We have constructed a signal pathway in bacteria for sensing the indicating-molecule Nitric Oxide of Inflammatory Bowel Disease and secreting Epidermal Growth Factors which contribute to the recovery of IBD. The pathway, which is illustrated in Figure A, consists of two sections: Nitric oxide sensing and Epidermal Growth Factor expression.</div>
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                        <p>As the first discovered cellular signaling molecule, Nitric Oxide (NO) plays a vital role in in many physiological and pathological processes. There’s a significant increase of NO concentration within the gut of IBD patients. <sup>[1-3]</sup> We utilized NO molecule as a “marker” for inflammatory position, and engineered E.coli to detect NO, so as to sense the inflammation.</p>
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                        <h3 id="NO_gradient_in_IBD_patients">NO gradient in IBD patients</h3>
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                                    <img class="img-responsive" style="margin:30px auto;" src="https://static.igem.org/mediawiki/2016/0/0d/ShanghaiTech_B_NO6.png">
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                        <p>Previous research showed that there’s a NO gradient within the gut of IBD patients. [1-3] The half-life of NO under physiological condition is only few seconds, so the NO gradient has a short distance but a sharp NO concentration peak at the inflammatory position. These features makes NO molecule a novel inflammation “marker”.</p>
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                        <cite>(此处补一张自制NO gradient的图，突出半衰期短，峰尖的特点……)</cite>
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                         <div class="p"><b>Click <a href="https://2016.igem.org/Team:ShanghaiTechChina_B/Project#Intestinal_Disease">here</a> to view EGF Expression</b></div>
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                         <h3 id="SNP_releasing_NO">SNP releasing NO</h3>
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                         <div class="p"><b>Click <a href="https://2016.igem.org/Team:ShanghaiTechChina_B/Project#NO_Sensor">here</a> to view NO Sensor</b></div>
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                        <p>There are several reagents that can release NO in aqueous solution. One of them is called <b>Sodium nitroprusside</b> (SNP), a member of the NO-releasing drugs, which is originally used as a peripheral vasodilator.</p>
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                        <p>In order to understand the kinetics of SNP releasing NO in LB, or say the environment where E.coli is cultured, we detected the concentration of NO in SNP-LB solution by Griess reagent.[4] And plotted the Dynamic curves of different concentrations of SNP releasing NO.</p>
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                         <div class="p">We have constructed a biofilm-display system. Beneficial molecules are linked to biofilm monomers to assembly outside the cell, forming extracellular matrix (Figure B). This would show the potential value of this display approach.</div>
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                            <img src="https://static.igem.org/mediawiki/2016/3/37/ShanghaiTech_B_NO_fig1.png" style="width:100%;">
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                            <p>Figure 1. The Dynamic curves of different concentrations of SNP releasing NO. The SNP concentrations are 10 μM, 100 μM, 1 mM, 10 mM and 0 μM (control) in each group. Data are means(SD) (n=3 samples per group).</p>
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                         <div class="p"><b>Click <a href="https://2016.igem.org/Team:ShanghaiTechChina_B/Project#Therapeutic_Biofilm">here</a> to view Therapeutic Biofilm</b></div>
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                         <p>The result shows the kinetics of SNP releasing NO in LB. From the dynamic curves, we could see that even low concentration of SNP could continuously release NO for 5~6 hours or even more, and the concentration of released NO is just appropriate for mimic the rectal NO concentration of patients of IBD, which is about 10 μM <sup>[3]</sup>.</p>
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                         <div class="p">We have constructed a kill switch which inhibits the growth of bacteria under our control (Figure C). This switch ensures the safety for both our lab work and the therapy.</div>
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                            <img src="https://static.igem.org/mediawiki/2016/7/70/ShanghaiTech_B_NO_fig2.png" style="width:100%;">
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                                    <img class="img-responsive" style="margin:30px auto;" src="https://static.igem.org/mediawiki/2016/e/e4/ShanghaiTech_Kill.png">
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                            <p>Figure 2. The concentration of NO released by SNP in LB medium were determined by Griess reagent. Add Griess reagents to samples within 96-well plate and read the absorbance at 540 nm by the microplate photometer.</p>
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                        <div class="p"><b>Click <a href="https://2016.igem.org/Team:ShanghaiTechChina_B/Project#Kill_Switch">here</a> to view Kill Switch</b></div>
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                        <p>These conclusions suggest that SNP releasing NO in LB culture could mimic the NO gradient in the inflammatory gut of IBD patients. (换个更好的用词)</p>
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                        <h3 id="NO_Sensing">NO Sensing</h3>
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                        <p>Microbiome have evolved several biological NO sensors, including SoxR protein, FNR family, NorR protein and so on. [4] NorR protein has an N-terminal regulatory domain, which is regulated by NO molecule, and a C-terminal DNA binding domain, which will binds to promoter pNorV to initiate transcription when activated.</p>
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                        <p>(此处补一张NorR，pNorV机理的图)</p>
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                        <p>We integrated this system to a vector into E.coli, which would produce NorR protein constitutively and activate pNorV transcription if NO exists.</p>
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                        <p>(此处脑补上构建成功的NNG图谱….-_-||)</p>
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                        <h4><b>Reference</b></h4>
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                         <p>[1] Rachmilewitz D, Stamler J S, Bachwich D, et al. Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease[J]. Gut, 1995, 36(5): 718-723.</p>
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                        <p>[2] Kimura H, Miura S, Shigematsu T, et al. Increased nitric oxide production and inducible nitric oxide synthase activity in colonic mucosa of patients with active ulcerative colitis and Crohn's disease[J]. Digestive diseases and sciences, 1997, 42(5): 1047-1054.</p>
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                        <p>[3] Ljung T, Herulf M, Beijer E, et al. Rectal nitric oxide assessment in children with Crohn disease and ulcerative colitis. Indicator of ileocaecal and colorectal affection[J]. Scandinavian journal of gastroenterology, 2001, 36(10): 1073-1076.</p>
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                        <p>[4] Nagano T. Practical methods for detection of nitric oxide[J]. Luminescence, 1999, 14(6): 283-290.</p>
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                        <p>[5] Spiro S. Regulators of bacterial responses to nitric oxide[J]. FEMS microbiology reviews, 2007, 31(2): 193-211.</p>
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                            <li><a href="#Introduction">Introduction</a></li>
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                            <li><a href="#NO_gradient_in_IBD_patients">NO gradient in IBD patients</a></li>
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                            <li><a href="#SNP_releasing_NO">SNP releasing NO</a></li>
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                            <li><a href="#NO_Sensing">NO Sensing</a></li>
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Latest revision as of 00:29, 20 October 2016

We have achieved following experiment results as our PROOF OF CONCEPT.
We have constructed a signal pathway in bacteria for sensing the indicating-molecule Nitric Oxide of Inflammatory Bowel Disease and secreting Epidermal Growth Factors which contribute to the recovery of IBD. The pathway, which is illustrated in Figure A, consists of two sections: Nitric oxide sensing and Epidermal Growth Factor expression.
Click here to view EGF Expression
Click here to view NO Sensor
We have constructed a biofilm-display system. Beneficial molecules are linked to biofilm monomers to assembly outside the cell, forming extracellular matrix (Figure B). This would show the potential value of this display approach.
Click here to view Therapeutic Biofilm
We have constructed a kill switch which inhibits the growth of bacteria under our control (Figure C). This switch ensures the safety for both our lab work and the therapy.
Click here to view Kill Switch
  • iGEM2016
  • Copyright © 2016 ShanghaiTechChina_B iGEM Team