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

 
Line 1: Line 1:
 
{{ShanghaiTechChina B}}
 
{{ShanghaiTechChina B}}
 
<html>
 
<html>
 
 
         <div class="container">
 
         <div class="container">
 
             <div class="row">
 
             <div class="row">
 
                 <div class="col-md-12">
 
                 <div class="col-md-12">
 
                     <div class="bs-docs-section">
 
                     <div class="bs-docs-section">
                         <div class="h1">Description</div>
+
                         <div class="h1"">We have achieved following experiment results as our PROOF OF CONCEPT.</div>
                         <div class="p">This year, we mainly tested two different kinds of killer genes, and turned to characterize them under different expression circuits.</div>
+
                         <div class="p">(A) We have constructed a signal pathway (in principle) in which bacteria detects Nitric Oxide (NO) released in the inflammation sites in IBD patients, expresses therapeutic molecule - Epidermal Growth Factors (EGF) and releases them back to inflammation sites to relieve IBD symptoms. This is illustrated in Figure 1. </div>
 +
                            <div class="row">
 +
                                <div class="col-md-12">
 +
                                    <img class="img-responsive" style="margin:30px auto;" src="https://static.igem.org/mediawiki/2016/0/0d/ShanghaiTech_B_NO6.png">
 +
                                </div>
 +
                                <div style="font-size:16px;text-align:center;width:80%;display:block;margin:10%;margin-bottom:1em;">
 +
                                    <b>Figure 1.</b>
 +
                                </div>
 +
                            </div>
 +
                        <div class="p">Specifically, 1) we mimicked NO release at pathological concentration by SNP (see <a href="https://2016.igem.org/Team:ShanghaiTechChina_B/Project#NO_Sensor">NO release</a>).</div>
 +
                        <div class="p">2) we successfully used NO (released by SNP) to induce expression of a demo protein (GFP), under control of a NO sensitive promoter (pNorV) (see <a href="https://2016.igem.org/Team:ShanghaiTechChina_B/Project#NO_Sensor">NO sensing</a>). The demo protein can be easily replaced by the therapeutic molecule – EGF.</div>
 +
                        <div class="p">3) we successfully demonstrated that our bacterial expressed EGF could relieve the symptoms in IBD mice (see <a href="https://2016.igem.org/Team:ShanghaiTechChina_B/Collaborations">collaboration</a>). EGF suppressed the reduction of colon length and rescued weight loss phenotype. </div>
  
                         <div class="p">During our experiment, we compared the property of killer red with gene E, both of them were linked to the same T7 promoter and RBS. At the same time, we tested the efficiency of gene E in different plasmid: pet28a and pBV220. (For the patent issues, we do not submit the temperature sensitive operon with gene E, but we insert the gene E into the enzyme cutting site of EcoRI and PstI, and anyone could use pBV220 to test our results.) </div>
+
                         <div class="p">(B) We have constructed a therapeutic biofilm system (in principle) for potential delivery of therapeutic molecules, in which we successfully fused a demo protein (EGF) to the biofilm protein CsgA in CsgA null bacteria (see <a href="https://2016.igem.org/Team:ShanghaiTechChina_B/Project#Therapeutic_Biofilm">therapeutic biofilm</a>). The demo protein can be easily replaced by other therapeutic molecules which can function remotely (such as enzymes) without detaching from the biofilm, and thereby helping patients in long term. This is illustrated in Figure 2.</div>
                        <div class="p">Induced by IPTG, E. coli transformed with killer red and gene E showed different behaviors. Under green laser, red fluorescence could be observed in induced E. coli which contained killer red. We got perfect image of red fluorescent by using confocal microscopy. By experiment, killer red could not be activated by light of shaker, but easily activated by green laser of fluorescence microscope. Using spread-plant method we tested the efficiency of killer red and got positive results. As for gene E, we made the subtraction of OD 600 and seen the slope as the index which show the effects of our gene E. </div>
+
                        <div class="p">Compared with gene E, killer red is more soft and safe. Based on the result, gene E tended to show property of leaking expression but killer red showed no toxin effect without either IPTG or green laser. Also, we could conclude from the data that gene E, linked with T7 promoter, restraining the growth of transformed E.coli, seemed more effective than killer red. </div>
+
                        <div class="p">When it comes to gene E, efficiency of it under different promoters are tested by OD 600 which indicated the concentration of E. coli. We made subtraction of OD 600 and saw the slope as the index which show the effects of our gene E. According the slope, we could found the efficiency of Gene E under T7 promoter is between the efficiency of gene E in pBV220 induced in 37℃ and that in 42℃. </div>
+
 
                             <div class="row">
 
                             <div class="row">
 
                                 <div class="col-md-12">
 
                                 <div class="col-md-12">
                                     <img class="img-responsive" style="margin:30px auto;" src="https://static.igem.org/mediawiki/2016/d/d0/ShanghaiTech_Kill14.png">
+
                                     <img class="img-responsive" style="margin:30px auto;" src="https://static.igem.org/mediawiki/2016/a/ad/ShanghaiTech_Biofilm2.png">
                                    <div style="font-size:16px;text-align:center;width:80%;display:block;margin:10%;margin-bottom:1em;margin-top:1em;">
+
                                </div>
                                     <b>Figure 1.</b> Efficiency of gene E in pBV220 induced in different temperatures
+
                                <div style="font-size:16px;text-align:center;width:80%;display:block;margin:10%;margin-bottom:1em;">
 +
                                     <b>Figure 2.</b>
 
                                 </div>
 
                                 </div>
                                </div>                           
 
 
                             </div>
 
                             </div>
 +
                        <div class="p">(C). We have constructed a “warship” to protect and confine our engineered bacteria, attempting to increase the surviving power of introduced bacteria against native microbiota and biosafety levels of using engineered bacteria, but still allow exchanging of nutrient and chemical through semi-permeable membranes (see <a href="https://2016.igem.org/Team:ShanghaiTechChina_B/Project#Warship">warship</a>). This is illustrated in Figure 3.</div>
 
                             <div class="row">
 
                             <div class="row">
 +
                                <div class="col-md-6">
 +
                                    <img class="img-responsive" style="height:300px;margin:30px auto;" src="https://static.igem.org/mediawiki/2016/e/e5/ShanghaiTech_B_Warship1.jpg">
 +
                                </div>
 +
                                <div class="col-md-6">
 +
                                    <img class="img-responsive" style="height:300px;margin:30px auto;" src="https://static.igem.org/mediawiki/2016/8/88/ShanghaiTech_B_Warship2.png">
 +
                                </div>
 +
                                <div style="font-size:16px;text-align:center;width:80%;display:block;margin:10%;margin-bottom:1em;">
 +
                                    <b>Figure 3.</b>
 +
                                </div>
 +
                            </div>
 +
 +
                        <div class="p">(D). We have constructed two kill switches which can inhibit bacteria growth under our control (see <a href="https://2016.igem.org/Team:ShanghaiTechChina_B/Project#Kill_Switch">kill switch</a>). We can either use light (via toxin fluorescent protein <a href="https://2016.igem.org/Team:ShanghaiTechChina_B/Project#Kill_Switch">killer red</a>) or a small molecule (via cell lysis protein <a href="https://2016.igem.org/Team:ShanghaiTechChina_B/Project#Kill_Switch">gene E</a>) to switch on the kill switch to terminate them when necessary. This is illustrated in Figure 4.</div>
 +
                        <div class="row>"
 
                                 <div class="col-md-12">
 
                                 <div class="col-md-12">
                                     <img class="img-responsive" style="margin:30px auto;" src="https://static.igem.org/mediawiki/2016/d/da/ShanghaiTech_Kill16.png">
+
                                     <img class="img-responsive" style="margin:30px auto;" src=" https://static.igem.org/mediawiki/2016/e/e8/ShanghaiTech_Proof.png">
                                    <div style="font-size:16px;text-align:center;width:80%;display:block;margin:10%;margin-bottom:1em;margin-top:1em;">
+
                                    <b>Figure 2.</b> Efficiency of gene in pet28 induced by 0.5 mM IPTG
+
 
                                 </div>
 
                                 </div>
                                 </div>                           
+
                                 <div style="font-size:16px;text-align:center;width:80%;display:block;margin:10%;margin-bottom:1em;">
                            </div>                           
+
                                    <b>Figure 4.</b>
                      <div class="p">It is quite pity that we did not have enough time to compare the efficiency of gene E and killer red quantitatively, we would continue our experiment and go into more detail of these two parts in the future.</div>
+
                                </div>
                      <div class="p">For more information about our description, please click this link: <a href="https://2016.igem.org/Team:ShanghaiTechChina_B/Project#Kill_Switch">Kill_Switch</a></div>
+
                        </div>
 +
 
 +
 
 +
 
 
                 </div>
 
                 </div>
 
             </div>
 
             </div>

Latest revision as of 03:38, 20 October 2016

We have achieved following experiment results as our PROOF OF CONCEPT.
(A) We have constructed a signal pathway (in principle) in which bacteria detects Nitric Oxide (NO) released in the inflammation sites in IBD patients, expresses therapeutic molecule - Epidermal Growth Factors (EGF) and releases them back to inflammation sites to relieve IBD symptoms. This is illustrated in Figure 1.
Figure 1.
Specifically, 1) we mimicked NO release at pathological concentration by SNP (see NO release).
2) we successfully used NO (released by SNP) to induce expression of a demo protein (GFP), under control of a NO sensitive promoter (pNorV) (see NO sensing). The demo protein can be easily replaced by the therapeutic molecule – EGF.
3) we successfully demonstrated that our bacterial expressed EGF could relieve the symptoms in IBD mice (see collaboration). EGF suppressed the reduction of colon length and rescued weight loss phenotype.
(B) We have constructed a therapeutic biofilm system (in principle) for potential delivery of therapeutic molecules, in which we successfully fused a demo protein (EGF) to the biofilm protein CsgA in CsgA null bacteria (see therapeutic biofilm). The demo protein can be easily replaced by other therapeutic molecules which can function remotely (such as enzymes) without detaching from the biofilm, and thereby helping patients in long term. This is illustrated in Figure 2.
Figure 2.
(C). We have constructed a “warship” to protect and confine our engineered bacteria, attempting to increase the surviving power of introduced bacteria against native microbiota and biosafety levels of using engineered bacteria, but still allow exchanging of nutrient and chemical through semi-permeable membranes (see warship). This is illustrated in Figure 3.
Figure 3.
(D). We have constructed two kill switches which can inhibit bacteria growth under our control (see kill switch). We can either use light (via toxin fluorescent protein killer red) or a small molecule (via cell lysis protein gene E) to switch on the kill switch to terminate them when necessary. This is illustrated in Figure 4.
Figure 4.