Difference between revisions of "Team:SZU-China/Design"

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                         <p style="font-size: 16px;">As has been described in the <a href="https://2017.igem.org/Team:SZU-China/Description" class="outlink" style="font-size:16px;font-weight:500">background</a>, we have to construct a self-repairing device in the concrete, which is achieved by genetic modified Bacillus subtilis embedded inside by micro-capsule.</p><br>
+
                         <p style="font-size: 16px;">As we have described in the <a href="https://2017.igem.org/Team:SZU-China/Description" class="outlink" style="font-size:16px;font-weight:500">background</a>, a self-repairing device in the concrete, which is achieved by genetic modified Bacillus subtilis embedded inside, is required.</p><br>
−
                         <p style="font-size: 16px;">To construct the bacteria properly functioning in the concrete, we should first improve the alkali resistance, germination rate and mineralization capacity of Bacillus subtilis significantly. Then, we use the special technology to make the spore of Bacillus subtilis into microcapsules, while adding the necessary nutrients for germination together. Finally we will incorporate the microcapsule into concrete in a certain proportion.</p><br>
+
                         <p style="font-size: 16px;">To ensure that bacteria can function properly in concrete, we first improved alkali resistance ability, germination ability and mineralization capacity of Bacillus subtilis significantly. Then, we used a special technique to make microcapsules containing spores of Bacillus subtilis, along with necessary nutrients for germination. The microcapsules are incorporated into concrete of a certain proportion.</p><br>
−
                         <p style="font-size: 16px;">When the microcracks inside the building appears, the microcapsules are torn and the water is infiltrated to dissolve the nutrient, which stimulates the spore into the germination state and finally returns to normal activity. Carbonic anhydrase of Bacillus subtilis promotes CO<sub>2</sub> hydration to produce CO<sub>3</sub><sup>2-</sup>, which binds to free Ca + in the environment to form calcium carbonate deposits, thereby enabling microcracks to self-repair and isolate the external environment from further corrosion of the internal reinforcement.</p>
+
                         <p style="font-size: 16px;">When microcracks inside buildings appear, the microcapsules will be torn apart and water is infiltrated to dissolve the nutrient. Spores are stimulated by mutrients into germination state and finally return to normal activity. Carbonic anhydrase of Bacillus subtilis promotes CO2 hydration to produce CO32-, which binds to free Ca2+ in the environment to form calcium carbonate deposits, thereby enabling microcracks to self-repair. As a result, microcracks are isolated from the external environment, preventing further corrosion due to internal reinforcement.</p>
  
 
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                     <div style=" font-size:25px;float:left;font-weight:500">WB800</div><p style="font-size: 16px;"> &nbsp; &nbsp;The chassis we chose this year is Bacillus subtilis because of its ability to form spore, which enable the bacteria to lie dormant for extended periods and protect itself from the harsh environment, like extreme pH, high temperature, and desiccation. In all the strains of Bacillus Subtilis, B.S168 is the most common one. However, since this strain secrete the digestive enzyme which will certainly interrupt the exogenous gene expression, we choose its modified strain – WB800, which has no digestive enzyme.</p>
+
                     <div style=" font-size:25px;float:left;font-weight:500">WB800</div><p style="font-size: 16px;"> &nbsp; &nbsp;We chose Bacillus subtilis this year as the chassis due to its ability to form spore, which enables the bacteria to lie dormant for extended periods and protect itself from harsh environment, for instant extreme high or low pH, high temperature, and desiccation. In all the strains of Bacillus Subtilis, B.S168 is the most common one. However, since Bacillus Subtilis of this strain secretes digestive enzymes, which interrupt exogenous gene expression, we chose a modified strain – WB800, which has no digestive enzymes.</p>
 
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                     <div style=" font-size:25px;float:left;font-weight:500">MCC micro-capsule</div><p style="font-size: 16px;">&nbsp; &nbsp;To allow WB800 properly function in the concrete to fill the crack, we have to ensure the bacteria remain as spore – a stable and dormant state in which it will remain uninterrupted by other factors until the cracks appearing. We need to insulate them from outside environment and revive them when cracks appear. Under this consideration, we design a kind of micro-capsule composed of MCC material sheltering the spore from outside. The MCC, short for microcrystalline cellulose, as a stable and non-toxic composite material, can certainly ensure the insolation between spores and outer environments. In the meantime, the use of micro-capsule can gather substantial spores together, thus promoting the spores’ concentration each unit volume and enhance the repairing effect. So we design the micro-capsule to properly embed the bacteria as spores in the concrete.</p>
+
                     <div style=" font-size:25px;float:left;font-weight:500">MCC micro-capsule</div><p style="font-size: 16px;">&nbsp; &nbsp;To allow WB800 function properly in concrete to fill the crack, we have to ensure that bacterium remain as sporse – a stable and dormant state in which it will remain uninterrupted by other factors until the cracks appear. Thus we need to insulate them from outside environment. Under this consideration, we designed a kind of micro-capsule composed of MCC material sheltering the spore from outside. MCC-short for microcrystalline cellulose-is a stable and non-toxic composite material, keeps spores in isolation from outer environment. In the meantime, use of micro-capsule can enwrap the spores, thus promoting the spores’ concentration in each unit volume and enhancing the repairing effect. So we designed the micro-capsule to embed bacterium as spores in concrete properly.</p>
 
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                 <span style="font-size: 36px;color: #4B97A5;">What function will the bacteria perform?</span>
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                 <span style="font-size: 36px;color: #4B97A5;">How will the bacteria perform?</span>
 
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                         <p style="font-size: 20px;color: #4B97A5;padding: 0">To activate the spore for germination</p>
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                         <p style="font-size: 20px;color: #4B97A5;padding: 0">To activate germination ability of spores</p>
−
                         <p style="font-size: 16px;">As mentioned above, the embedded spore remains dormant in the micro-capsule. In order to wake them up for function, we transferred the gene gerA into our bacteria and overexpress it. This gene encodes the nutrient receptor on the inner membrane which can detect the outer nutrient substance for example, L-alanine, and activates the germination and initiate our system through complex signaling pathways.</p>
+
                         <p style="font-size: 16px;">As mentioned above, the embedded spores remain dormant in the micro-capsule. In order to wake them up, we transferred the gene, gerA, into our bacteria and overexpressed it. This gene encodes the nutrient receptor on the inner membrane, which can detect the outer nutrient substance, for example L-alanine, and activate germination and initiate our system through complex signaling pathways.</p>
  
 
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                     <p style="font-size: 20px;color: #4B97A5;padding: 20px 0">To resist certain alkaline environment</p>
 
                     <p style="font-size: 20px;color: #4B97A5;padding: 20px 0">To resist certain alkaline environment</p>
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                     <p style="font-size: 16px;">Considering the working circumstance of our system is concrete which has rather high pH, We need to enhance the alkali resistance of our bacteria. Thus we transferred two alkali resistant genes.</p>
+
                     <p style="font-size: 16px;">Considering the working circumstance of our system, concrete, where pH level is rather high, we need to enhance the alkali resistance ability of our bacteria. Thus we transferred two alkali resistant genes.</p>
−
                     <p style="font-size: 14px;padding: 20px 0 20px 20px">1. The expression of gene tupA is a kind of enzyme which can shift glucuronic acid and L-glutamic acid into polyglucuronic acid and poly-γ-L-glutamic acid. These polymers will form a layer of protection barrier on the cell wall and neutralize the extracellular hydroxyl. </p>
+
                     <p style="font-size: 14px;padding: 20px 0 20px 20px">1. The expression of gene tupA results in a kind of enzyme which can shift glucuronic acid and L-glutamic acid into polyglucuronic acid and poly-γ-L-glutamic acid. These polymers will form a layer of protection barrier on the cell wall and neutralize the extracellular hydroxyl. </p>
−
                     <p style="font-size: 14px;padding: 20px 0 20px 20px">2. In the meantime we transferred the second alkali resistant gene, nhaC. It is the coding gene of the proton pump on the cell membrane. And this specific proton pump play a key role in regulating cytoplasmic pH value by coupling net hydrogen ions in and sodions out simultaneously, thus can balance the endocellular pH.</p>
+
                     <p style="font-size: 14px;padding: 20px 0 20px 20px">2. In the meantime, we transferred the second alkali resistant gene, nhaC. It is the coding gene for proton pumps on the cell membrane. These proton pumps play a key role in regulating cytoplasmic pH valued by coupling net hydrogen ions in and sodions out simultaneously, thus can balance the endocellular pH.</p>
  
 
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                         <p style="font-size: 20px;color: #4B97A5;padding: 20px 0">To produce CaCO<sub>3</sub> for crack filling</p>
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                         <p style="font-size: 20px;color: #4B97A5;padding: 20px 0">To produce CaCO<sub>3</sub> for crack-filling</p>
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                         <p style="font-size: 16px;">The key point of self-repairing system is to allow our bacteria producing calcium carbonate for crack filling. In order to realize this, we transferred the gene of carbonic anhydrase. It is a highly efficient enzymes catalyzes the reversible hydration of CO<sub>2</sub> and forming bicarbonate and protons rapidly. The bicarbonate radicals will diffuse out of the cell along with the concentration gradient and combine with the hydroxyl and calcium iron in the concrete environment, which will become calcium carbonate sediment. Now the calcium carbonate can go and fill the micro cracks.</p>
+
                         <p style="font-size: 16px;">The key point of self-repairing system is to allow our bacteria producing calcium carbonate for crack-filling. To achieve this goal, we transferred the gene of carbonic anhydrase into our bacteria. This gene produces highly efficient enzymes that catalyze the reversible reaction of hydration of CO2 and form bicarbonate and protons rapidly. bicarbonate radicals will diffuse out of the cell along with the concentration gradient and combine with the hydroxyl and calcium iron in the concrete environment, which will become calcium carbonate sediment. Now the calcium carbonate can go and fill the micro cracks.</p>
 
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Revision as of 10:50, 1 November 2017

DESIGN

As we have described in the background, a self-repairing device in the concrete, which is achieved by genetic modified Bacillus subtilis embedded inside, is required.


To ensure that bacteria can function properly in concrete, we first improved alkali resistance ability, germination ability and mineralization capacity of Bacillus subtilis significantly. Then, we used a special technique to make microcapsules containing spores of Bacillus subtilis, along with necessary nutrients for germination. The microcapsules are incorporated into concrete of a certain proportion.


When microcracks inside buildings appear, the microcapsules will be torn apart and water is infiltrated to dissolve the nutrient. Spores are stimulated by mutrients into germination state and finally return to normal activity. Carbonic anhydrase of Bacillus subtilis promotes CO2 hydration to produce CO32-, which binds to free Ca2+ in the environment to form calcium carbonate deposits, thereby enabling microcracks to self-repair. As a result, microcracks are isolated from the external environment, preventing further corrosion due to internal reinforcement.

Which chassis to choose?
WB800

   We chose Bacillus subtilis this year as the chassis due to its ability to form spore, which enables the bacteria to lie dormant for extended periods and protect itself from harsh environment, for instant extreme high or low pH, high temperature, and desiccation. In all the strains of Bacillus Subtilis, B.S168 is the most common one. However, since Bacillus Subtilis of this strain secretes digestive enzymes, which interrupt exogenous gene expression, we chose a modified strain – WB800, which has no digestive enzymes.

How to embed the bacteria into the concrete?
MCC micro-capsule

   To allow WB800 function properly in concrete to fill the crack, we have to ensure that bacterium remain as sporse – a stable and dormant state in which it will remain uninterrupted by other factors until the cracks appear. Thus we need to insulate them from outside environment. Under this consideration, we designed a kind of micro-capsule composed of MCC material sheltering the spore from outside. MCC-short for microcrystalline cellulose-is a stable and non-toxic composite material, keeps spores in isolation from outer environment. In the meantime, use of micro-capsule can enwrap the spores, thus promoting the spores’ concentration in each unit volume and enhancing the repairing effect. So we designed the micro-capsule to embed bacterium as spores in concrete properly.

How will the bacteria perform?

To activate germination ability of spores

As mentioned above, the embedded spores remain dormant in the micro-capsule. In order to wake them up, we transferred the gene, gerA, into our bacteria and overexpressed it. This gene encodes the nutrient receptor on the inner membrane, which can detect the outer nutrient substance, for example L-alanine, and activate germination and initiate our system through complex signaling pathways.

To resist certain alkaline environment

Considering the working circumstance of our system, concrete, where pH level is rather high, we need to enhance the alkali resistance ability of our bacteria. Thus we transferred two alkali resistant genes.

1. The expression of gene tupA results in a kind of enzyme which can shift glucuronic acid and L-glutamic acid into polyglucuronic acid and poly-γ-L-glutamic acid. These polymers will form a layer of protection barrier on the cell wall and neutralize the extracellular hydroxyl.

2. In the meantime, we transferred the second alkali resistant gene, nhaC. It is the coding gene for proton pumps on the cell membrane. These proton pumps play a key role in regulating cytoplasmic pH valued by coupling net hydrogen ions in and sodions out simultaneously, thus can balance the endocellular pH.

To produce CaCO3 for crack-filling

The key point of self-repairing system is to allow our bacteria producing calcium carbonate for crack-filling. To achieve this goal, we transferred the gene of carbonic anhydrase into our bacteria. This gene produces highly efficient enzymes that catalyze the reversible reaction of hydration of CO2 and form bicarbonate and protons rapidly. bicarbonate radicals will diffuse out of the cell along with the concentration gradient and combine with the hydroxyl and calcium iron in the concrete environment, which will become calcium carbonate sediment. Now the calcium carbonate can go and fill the micro cracks.

Here is a video for the specific mechanism described above.

Reference

Microcrystalline celluloseMicrocrystalline celluloseMicrocrystalline celluloseMicrocrystalline celluloseMicrocrystalline celluloseMicrocrystalline celluloseMicrocrystalline cellulose