Difference between revisions of "Team:ECUST/Demonstrate"

 
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<div id="pagebanner" style="background-image:url(https://static.igem.org/mediawiki/2018/4/41/T--ECUST--demonstrationbanner.png);">
 
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    <div id="bannerquote">DEMONSTRATION</div>
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      <div id="bannerquote">Demonstrate</div>
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<main>
 
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<h1 class="box-heading">Design</h1>
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<h1 class="box-heading">CULTURE AND IDENTIFICATION OF IRON BACTERIA  </h1>
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<figure>
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<figure class="makeresponsive" style="width: 50%;">
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<img src="https://static.igem.org/mediawiki/2018/6/64/T--ECUST--Dd1.jpg" class="z1oom">
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<figcaption><b>Figure 1. (a)Bacteria is screened from rusty iron sample and cultured with Winogradsky culture medium. With rust and biofilm, bacterial colony is bronzing and circular. (b)Gram Straing：Gram-negative bacteria. (c)Iron bacteria with its biofilm (d)Electron microscope picture of iron bacteria with rust and biofilm complex.</b></figcaption>
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<p>
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<figure>
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Since the current solution to the blockage is not environmentally friendly and economical, we hope to use synthetic biology to solve the three problems of rust deposition, biofilm accumulation and microbial activity by engineering microbe.
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</p>
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<img src="https://static.igem.org/mediawiki/2018/d/d0/T--ECUST--D2.jpg" class="z2oom">
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<figcaption><b>Figure 2. The iron bacteria belongs to Pseudomonas sp. According to the identification results of 16s rDNA</b></figcaption>
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<figure>
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However, the three functions of engineering bacteria may bring great metabolic pressure to the engineering bacteria, and the anti-bacterial substances produced by them may adversely affect their growth and reproduction. So we need to design a set of genetic circuit to improve this situation and achieve a sequential expression of these three functions!
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<img src="https://static.igem.org/mediawiki/2018/5/55/T--ECUST--D3.jpg" class="z3oom">
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<figcaption><b>Figure 3: Iron bacteria growth curve.Bacteria is cultured in Winogradsky culture medium at 30℃ and shake at 220rpm, 2% inoculum of culture solution (culturing age 3 days) and measure the light absorption at 600 nm.</b></figcaption>
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Inspired by the way people currently deal with it, people first add rust remover and biofilm remover, which first destroys the living environment of the microorganisms, causing them to fail to receive protection, and then adding bactericide to achieve high bactericidal effect. Therefore, in our gene circuit, it is necessary to perform the function of destroying the microenvironment of the microbial before performing the sterilization function.
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</p>
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</div>
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<div class="contentbox">
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<h1 class="box-heading">QUORUM SENSING  </h1>
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<p>We constructed a quorum-sensing system and found that the promoter with afe-box can work when the AHL concentration is more than 10<sup>-7</sup> mol/L. </p>
 
<figure>
 
<figure>
 
<figure class="makeresponsive" style="width: 50%;">
 
<figure class="makeresponsive" style="width: 50%;">
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<img src="https://static.igem.org/mediawiki/2018/c/c8/T--ECUST--design1.png" alt="Figure1.1 OD600 reference point tab" class="zoom">
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<img src="https://static.igem.org/mediawiki/2018/5/58/T--ECUST--D4.jpg" class="zo4om">
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<figcaption><b></b></figcaption>
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<figcaption><b>Figure 4. (a) Fluorescence intensity at different time induced by different concentrations of AHL.
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      (b)A three-dimensional map with log10 concentration of AHL,time and fluorescence intensity as the coordinate
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</b></figcaption>
 
</figure>
 
</figure>
  
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<p>
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<p><a target="_blank" style="color:white; text-decoration:underline;" href="https://2018.igem.org/Team:ECUST/Experiments"><i>Click here for detailed information.</i></a><p>
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To perform the function of destroying the microenvironment, the first thing is to sense it!
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For the identification of the bacterial microenvironment, we use quorum sensing to achieve it. This enables our engineered E. coli to recognize the quorum sensing signal molecule HSL produced by iron bacteria, activate the expression of biological rust removers and biological biofilm remover. The biological rust remover we chose is the strongest ferric chelating agent-siderophore in nature. As for biofilm remover we chose a glycosidase from the actinomycetemella actinomycete that degrades the biofilm polysaccharide backbone.
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</p>
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<h1 class="box-heading">RUST REMOVER </h1>
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<p>
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<p>We have not successfully constructed the recombinant plasmid with enterobactin because the gene cluster is too long.But we purified the enterobactin from E. coli successfully and tested the rust removal effect. </p>
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With the progress of rust removing, the siderophore-Fe3+ complex concentration will continue to increase, and we hope that after the bacteria sense this signal, the expression of the biocide will be initiated. But in the natural iron sensing system, high concentration of iron inhibit the expression of genes, so we need to modify them. By introducing the foreign gene- lacI, the lacI was inhibited under high-iron conditions, and lacI inhibited the biocide gene, thereby realizing the design of the iron reversal system.
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</p>
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<p>
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Biocides we tried to use cecropin AD, an antibacterial peptide derived from silkworm, which has a spectral and efficient bactericidal effect. In order to release this peptide to the extracellular, we achieve cell rupture by simultaneously expressing the autolysin gene.
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</p>
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<figure>
 
<figure>
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<figure class="makeresponsive" style="width: 70%;">
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<figure class="makeresponsive" style="width: 50%;">
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<img src="https://static.igem.org/mediawiki/2018/4/47/T--ECUST--design3.png" alt="Figure1.1 OD600 reference point tab" class="zoom">
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<img src="https://static.igem.org/mediawiki/2018/4/46/T--ECUST--D5.jpg" class="zoo5m">
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<figcaption><b></b></figcaption>
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<figcaption><b>Figure 5. Fitting curves of oxalic acid and enterobactin
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(a) 3D fitting figure of rust removal rate for oxalic acid ;(b) 3D fitting figure of rust removal rate for enterobactin; (c) fitting curve of rust removal rate for oxalic acid ;(b) fitting curve of rust removal rate for enterobactin.
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</b></figcaption>
 
</figure>
 
</figure>
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<p>
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<p><a target="_blank" style="color:white; text-decoration:underline;" href="https://2018.igem.org/Team:ECUST/Rust_Remover"><i>Click here for detailed information.</i></a><p>
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For biosafety reasons, avoid engineering bacteria to escape from the piping system into the natural environment. We think of the dark environment inside the pipeline, and the natural environment is the light environment, so the light-on suicide is adopted, and the suicide gene is expressed when there is light to commit suicide.
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<p>
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For higher biosafety and detectability, we have designed a new hardware-iTube that can be applied to the piping system. ITube's built-in sensors can detect circulating water parameters, use artificial neural networks to conduct corrosion prediction and blockage assessment of cooling water systems, reminding people to add engineering bacteria for plugging and cleaning. At the same time, the built-in light system can also be manually turned on and off, makes the growth of the engineered bacteria in pipe is artificially regulated.
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</p>
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</div>
  
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<div class="contentbox">
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<h1 class="box-heading">BIOFILM REMOVER </h1>
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<p>We intesrt the gene of DispersinB (DSPB) to pET28a and E.coli expressed DSPB successfully.
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We performed enzyme activity assay and biofilm removal experiment by using cell supernatant. The enzyme activity of the supernatant is 66.363 U/mL. The supernatant has high biofilm removal rate, and the biofilm removal effect increased with time. </p>
 
<figure>
 
<figure>
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<figure class="makeresponsive floatleft" style="width: 50%;">
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<figure class="makeresponsive" style="width: 50%;">
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<img src="https://static.igem.org/mediawiki/2018/5/52/T--ECUST--design2.png" alt="Figure1.1 OD600 reference point tab" class="zoom">
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<img src="https://static.igem.org/mediawiki/2018/8/8c/T--ECUST--D6.jpg" class="zoo6m">
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<figcaption><b></b></figcaption>
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<figcaption><b>Figure 6. (a) Crystal violet staining results of different reaction solutions added to biofilm
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          (b) Biofilm removal rate of different reaction solutions added to biofilm
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          (c) DSPB biofilm-removing curve
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OD<sub>570</sub>=51.96-51.24t0.0051
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R<sup>2</sup>=0.98
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</b></figcaption>
 
</figure>
 
</figure>
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<p><a target="_blank" style="color:white; text-decoration:underline;" href="https://2018.igem.org/Team:ECUST/Biofilm_Remover"><i>Click here for detailed information.</i></a><p>
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</div>
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<div class="contentbox">
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<h1 class="box-heading">FUR INVERTER </h1>
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<p>The fur-box is inserted into three different positions of the promoter ,named Pfur,Pfur2 and Pfur.</p>
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<figure>
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<figure class="makeresponsive" style="width: 50%;">
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<img src="  https://static.igem.org/mediawiki/2018/f/f6/T--ECUST--D7.jpg
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" class="zoo7m">
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<figcaption><b> Figure 7.  (a) Relative expression of mCherry under the regulate of different fur box with Fe concentration of 10<sup>-5</sup>M 
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(b)Relative expression of mCherry under the regulate of different fur box with Fe concentration of 10<sup>-7</sup>M.
 +
Fe excess medium is used to measure expression of report gene.
 +
</b></figcaption>
 +
</figure>
 +
 +
<p>After adding with 10<sup>-7</sup>M Fe in medium ,Fur2 has the highest relative expression.So finally we choose fur2. </p>
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<p><a target="_blank" style="color:white; text-decoration:underline;" href="https://2018.igem.org/Team:ECUST/Fur_Inverter"><i>Click here for detailed information.</i></a><p>
  
 
</div>
 
</div>
  
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<div class="contentbox">
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<h1 class="box-heading">LYSIN </h1>
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<p>Recombinant E. coli successfully expressed Lysin and can can make holes in the cell membrane. </p>
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<figure>
 +
<figure class="makeresponsive" style="width: 50%;">
 +
<img src="https://static.igem.org/mediawiki/2018/c/c1/T--ECUST--D8.jpg" class="zoo8m">
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<figcaption><b> Figure 8. The  images are from scanning electron microscopes.(a)Before induction.(b)After induction.There are holes in the plasma membrane after induction, which can prove that lysin expresses successfully.
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</b></figcaption>
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</figure>
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<p><a target="_blank" style="color:white; text-decoration:underline;" href="https://2018.igem.org/Team:ECUST/Lysin"><i>Click here for detailed information.</i></a><p>
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</div>
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<div class="contentbox">
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<h1 class="box-heading">BIOCIDE </h1>
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<p>Recombinant E. coli successfully expressed  cecropin AD and we test the the sterilization effect of  cecropin AD . </p>
 +
<figure>
 +
<figure class="makeresponsive" style="width: 50%;">
 +
<img src=" https://static.igem.org/mediawiki/2018/b/ba/T--ECUST--D9.jpg" class="zoo9m">
 +
<figcaption><b> Figure 9. Growth curve of iron bacteria adding with different concentration of cecropin AD.</b></figcaption>
 +
</figure>
 +
<p>The figure shows that cecropin AD has powerful bacterividal activity. Only concentration of cecropin AD is low enough, can iron bacteria grow. </p>
 +
<p><a target="_blank" style="color:white; text-decoration:underline;" href="https://2018.igem.org/Team:ECUST/Biocide"><i>Click here for detailed information.</i></a><p>
 +
</div>
 +
 +
 +
<div class="contentbox">
 +
<h1 class="box-heading">LIGHT-ON SUICIDE </h1>
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<p>We test the growth curve of Recombinant E. coli and found that cell growth can be inhibited after light illumination. </p>
 +
 +
 +
<figure>
 +
<figure class="makeresponsive" style="width: 50%;">
 +
<img src=" https://static.igem.org/mediawiki/2018/a/ad/T--ECUST--D10.jpg" class="z10oom">
 +
<figcaption><b> Figure 10.  E.coil growth curve under different light conditions.
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</b></figcaption>
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</figure>
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<p><a target="_blank" style="color:white; text-decoration:underline;" href="https://2018.igem.org/Team:ECUST/Light-on_Suicide"><i>Click here for detailed information.</i></a><p>
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</div>
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<div class="contentbox">
 +
<h1 class="box-heading">IRON BACTERIA-KILL CONPREHENSIVE CHARACTERIZATION </h1>
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<p>Iron bacteria is cultured in Winogradsky culture medium with 1% inoculum size from seed medium.Then medium solution is transferred into 96-well microtiter plates with different culture conditions. Then bacteria is cultured for 24 hours and OD600 is measured. </p>
 +
<p>Effects of killing iron bacteria with Cecropin AD, Dsp B and enterobactin are considered comprehensively. </p>
 +
<p>Dsp B is crude enzyme solution from broken cell supernatant. </p>
 +
<p>Enterobacin is purified cell culture solution supernatant as what mentioned before. Cecropin AD is synthesized from Genscript. Pepide is dissolved in 1% acetic acid. </p>
 +
 +
<figure>
 +
<figure class="makeresponsive" style="width: 50%;">
 +
<img src="  https://static.igem.org/mediawiki/2018/b/ba/T--ECUST--D11.jpg" class="zo11om">
 +
<figcaption><b> Figure 11. Growth curve of iron bacteria adding with different concentration of cecropin AD and Dsp B.
 +
</b></figcaption>
 +
</figure>
 +
<p>From the picture, we can see that co-action of Dsp B and cecropin AD strengthens the effects of iron bacteria-killing. Dsp B can damage the environment iron bacteria live in, so antibacterial peptide can attack bacteria more easily. </p>
 +
<figure>
 +
<figure class="makeresponsive" style="width: 50%;">
 +
<img src="  https://static.igem.org/mediawiki/2018/0/01/T--ECUST--D12.jpg" class="zo12om">
 +
<figcaption><b> Figure 12. Growth curve of iron bacteria adding with different concentration of cecropin AD and enterobactin
 +
</b></figcaption>
 +
</figure>
 +
<p>We can see from the figure that co-action of cecropin AD and enterobactin can inhibit the growth of iron bacteria more effective. Enterobactin can despoil ferric iron in culture medium and cecropin can kill bacteria, so co-action of them can obviously kill iron bacteria. </p>
 +
<figure>
 +
<figure class="makeresponsive" style="width: 50%;">
 +
<img src="  https://static.igem.org/mediawiki/2018/1/17/T--ECUST--D13.jpg" class="z13oom">
 +
<figcaption><b> Figure 13.  Co-action with cecropin AD , enterobactin and Dsp B
 +
</b></figcaption>
 +
</figure>
 +
<p>We can see from the figure that adding with cecropin AD , DspB and enterobactin can kill iron bacteria powerfully. DspB and enterobactin can damage living environment of iron bacteria. When iron bacteria loses its biofilm and its energy source, bacteria can not survive. </p>
 +
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</div>
 
</main>
 
</main>
 
</div>
 
</div>

Latest revision as of 11:26, 2 November 2018

Demonstrate

CULTURE AND IDENTIFICATION OF IRON BACTERIA

Figure 1. (a)Bacteria is screened from rusty iron sample and cultured with Winogradsky culture medium. With rust and biofilm, bacterial colony is bronzing and circular. (b)Gram Straing：Gram-negative bacteria. (c)Iron bacteria with its biofilm (d)Electron microscope picture of iron bacteria with rust and biofilm complex.
Figure 2. The iron bacteria belongs to Pseudomonas sp. According to the identification results of 16s rDNA
Figure 3: Iron bacteria growth curve.Bacteria is cultured in Winogradsky culture medium at 30℃ and shake at 220rpm, 2% inoculum of culture solution (culturing age 3 days) and measure the light absorption at 600 nm.

QUORUM SENSING

We constructed a quorum-sensing system and found that the promoter with afe-box can work when the AHL concentration is more than 10-7 mol/L.

Figure 4. (a) Fluorescence intensity at different time induced by different concentrations of AHL. (b)A three-dimensional map with log10 concentration of AHL,time and fluorescence intensity as the coordinate

Click here for detailed information.

RUST REMOVER

We have not successfully constructed the recombinant plasmid with enterobactin because the gene cluster is too long.But we purified the enterobactin from E. coli successfully and tested the rust removal effect.

Figure 5. Fitting curves of oxalic acid and enterobactin (a) 3D fitting figure of rust removal rate for oxalic acid ;(b) 3D fitting figure of rust removal rate for enterobactin; (c) fitting curve of rust removal rate for oxalic acid ;(b) fitting curve of rust removal rate for enterobactin.

Click here for detailed information.

BIOFILM REMOVER

We intesrt the gene of DispersinB (DSPB) to pET28a and E.coli expressed DSPB successfully. We performed enzyme activity assay and biofilm removal experiment by using cell supernatant. The enzyme activity of the supernatant is 66.363 U/mL. The supernatant has high biofilm removal rate, and the biofilm removal effect increased with time.

Figure 6. (a) Crystal violet staining results of different reaction solutions added to biofilm (b) Biofilm removal rate of different reaction solutions added to biofilm (c) DSPB biofilm-removing curve OD570=51.96-51.24t0.0051 R2=0.98

Click here for detailed information.

FUR INVERTER

The fur-box is inserted into three different positions of the promoter ,named Pfur,Pfur2 and Pfur.

Figure 7. (a) Relative expression of mCherry under the regulate of different fur box with Fe concentration of 10-5M (b)Relative expression of mCherry under the regulate of different fur box with Fe concentration of 10-7M. Fe excess medium is used to measure expression of report gene.

After adding with 10-7M Fe in medium ,Fur2 has the highest relative expression.So finally we choose fur2.

Click here for detailed information.

LYSIN

Recombinant E. coli successfully expressed Lysin and can can make holes in the cell membrane.

Figure 8. The images are from scanning electron microscopes.(a)Before induction.(b)After induction.There are holes in the plasma membrane after induction, which can prove that lysin expresses successfully.

Click here for detailed information.

BIOCIDE

Recombinant E. coli successfully expressed cecropin AD and we test the the sterilization effect of cecropin AD .

Figure 9. Growth curve of iron bacteria adding with different concentration of cecropin AD.

The figure shows that cecropin AD has powerful bacterividal activity. Only concentration of cecropin AD is low enough, can iron bacteria grow.

Click here for detailed information.

LIGHT-ON SUICIDE

We test the growth curve of Recombinant E. coli and found that cell growth can be inhibited after light illumination.

Figure 10. E.coil growth curve under different light conditions.

Click here for detailed information.

IRON BACTERIA-KILL CONPREHENSIVE CHARACTERIZATION

Iron bacteria is cultured in Winogradsky culture medium with 1% inoculum size from seed medium.Then medium solution is transferred into 96-well microtiter plates with different culture conditions. Then bacteria is cultured for 24 hours and OD600 is measured.

Effects of killing iron bacteria with Cecropin AD, Dsp B and enterobactin are considered comprehensively.

Dsp B is crude enzyme solution from broken cell supernatant.

Enterobacin is purified cell culture solution supernatant as what mentioned before. Cecropin AD is synthesized from Genscript. Pepide is dissolved in 1% acetic acid.

Figure 11. Growth curve of iron bacteria adding with different concentration of cecropin AD and Dsp B.

From the picture, we can see that co-action of Dsp B and cecropin AD strengthens the effects of iron bacteria-killing. Dsp B can damage the environment iron bacteria live in, so antibacterial peptide can attack bacteria more easily.

Figure 12. Growth curve of iron bacteria adding with different concentration of cecropin AD and enterobactin

We can see from the figure that co-action of cecropin AD and enterobactin can inhibit the growth of iron bacteria more effective. Enterobactin can despoil ferric iron in culture medium and cecropin can kill bacteria, so co-action of them can obviously kill iron bacteria.

Figure 13. Co-action with cecropin AD , enterobactin and Dsp B

We can see from the figure that adding with cecropin AD , DspB and enterobactin can kill iron bacteria powerfully. DspB and enterobactin can damage living environment of iron bacteria. When iron bacteria loses its biofilm and its energy source, bacteria can not survive.