Difference between revisions of "Team:NCTU Formosa/Engineering"
| Line 12: | Line 12: | ||
<h1 class="topic" id="topic1">Research</h1> | <h1 class="topic" id="topic1">Research</h1> | ||
<p id="p1"> | <p id="p1"> | ||
| − |   From the research, we know that periodontal disease was proved to be related to many chronic and cardiovascular diseases. Among the pathogenic organisms of periodontal disease, <i>Porphyromonas gingivalis</i> is the most important bacteria that give rise to this bad condition in dogs. To tackle the problem at its roots, we put effort into finding the efficient way to reach the | + |   From the research, we know that periodontal disease was proved to be related to many chronic and cardiovascular diseases. Among the pathogenic organisms of periodontal disease, <i>Porphyromonas gingivalis</i> is the most important bacteria that give rise to this bad condition in dogs. To tackle the problem at its roots, we put effort into finding the efficient way to reach the inhibition of <i>Porphyromonas gingivalis</i> first. |
</p> | </p> | ||
<p id="p2"> | <p id="p2"> | ||
| − |   Finally, we chose antimicrobial peptides, LL-37 play the part of bacteria- | + |    Finally, we chose antimicrobial peptides, LL-37 play the part of bacteria-inhibition. LL-37 not only can inhibit <i>P. gingivalis</i> [1], can also inhibit <i>E. coli</i> itself [2], preventing DenTeeth from overgrowing after entering the canine mouth. |
</p> | </p> | ||
</div> | </div> | ||
| Line 22: | Line 22: | ||
<h1 class="topic" id="topic2">Design</h1> | <h1 class="topic" id="topic2">Design</h1> | ||
<p id="p1"> | <p id="p1"> | ||
| − |    Sterilization is the key to periodontal disease. For the wet lab, we first expect the LL-37 could attain the mission. To observe the expression of DenTeeth’s biobrick, the RFP would be inserted behind the LL37. Because | + |    Sterilization is the key to periodontal disease. For the wet lab, we first expect the LL-37 could attain the mission. To observe the expression of DenTeeth’s biobrick, the RFP would be inserted behind the LL37. Because DenTeeth would kill by LL-37 peptide too, we added a composite part in the front of the LL-37 producing sequence, preventing engineered bacteria died before entering the mouth. |
| + | </p> | ||
| + | <p> | ||
| + |    As stated before, LL-37 can inhibit E. coli. If the DenTeeth could not exist with LL-37 in a proper proportion, the DenTeeth wouldn't attain the mission of solving the periodontal disease. Therefore, we decided to use the prediction model to predict whether DenTeeth could grow and express under the effect of LL-37. To confirm the feasibility of DenTeeth. | ||
</p> | </p> | ||
<img src="https://static.igem.org/mediawiki/2021/4/45/T--NCTU_Formosa--biobrickAB.png" class="images" id=" Growth_P.gingivalis" alt=" growth curve of E. coli and P.gingivalis"/> | <img src="https://static.igem.org/mediawiki/2021/4/45/T--NCTU_Formosa--biobrickAB.png" class="images" id=" Growth_P.gingivalis" alt=" growth curve of E. coli and P.gingivalis"/> | ||
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d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"> | d="M8 256C8 119 119 8 256 8s248 111 248 248-111 248-248 248S8 393 8 256zm143.6 28.9l72.4-75.5V392c0 13.3 10.7 24 24 24h16c13.3 0 24-10.7 24-24V209.4l72.4 75.5c9.3 9.7 24.8 9.9 34.3.4l10.9-11c9.4-9.4 9.4-24.6 0-33.9L273 107.7c-9.4-9.4-24.6-9.4-33.9 0L106.3 240.4c-9.4 9.4-9.4 24.6 0 33.9l10.9 11c9.6 9.5 25.1 9.3 34.4-.4z"> | ||
</path> | </path> | ||
| − | </svg>Figure 1. | + | </svg>Figure 1. The biobrick of DenTeeth(A: 37°C Induced biobrick; B: LL-37 Producing Biobrick)</div> |
<p> | <p> | ||
  Only when the temperature is up than 37°C, the LuxR would be expressed (Fig.1 A). At this moment, if the concentration of bacteria is high, the amount of AHL would rise, binding with LuxR. The complex of AHL and LuxR will attach to the Plux (Fig.1 B), activating the expression of LL-37. |   Only when the temperature is up than 37°C, the LuxR would be expressed (Fig.1 A). At this moment, if the concentration of bacteria is high, the amount of AHL would rise, binding with LuxR. The complex of AHL and LuxR will attach to the Plux (Fig.1 B), activating the expression of LL-37. | ||
| Line 42: | Line 45: | ||
<div class="section s3"> | <div class="section s3"> | ||
<h1 class="topic" id="topic3">Build</h1> | <h1 class="topic" id="topic3">Build</h1> | ||
| − | + | <h2 class="subtopic">Cloning Result</h2> | |
<p id="p1"> | <p id="p1"> | ||
| − |   We successfully build engineering bacteria with LL-37 | + |   We successfully build engineering bacteria with LL-37. |
</p> | </p> | ||
<img class="images" style="width: 40%;" src="https://static.igem.org/mediawiki/2021/e/ea/T--NCTU_Formosa--LL-37_gel.jpg"> | <img class="images" style="width: 40%;" src="https://static.igem.org/mediawiki/2021/e/ea/T--NCTU_Formosa--LL-37_gel.jpg"> | ||
| Line 53: | Line 56: | ||
</path> | </path> | ||
</svg>Figure 2. Colony PCR result of LL-37 sequence after cloning into<i> E. coli </i>BL21. (1875 b.p.)</div> | </svg>Figure 2. Colony PCR result of LL-37 sequence after cloning into<i> E. coli </i>BL21. (1875 b.p.)</div> | ||
| − | |||
| − | |||
<p id="p2"> | <p id="p2"> | ||
  Through the modeling build with substituting parameters from published articles, the growth of <i>E. coli</i> and <i>P. gingivalis</i> under the effect of LL-37 would be inhibited significantly. However, the figure also show that <i>E. coli</i> could still live with LL-37. Simply speaking, we successfully test the feasibility of denteeth and find that we can improve the denteeth with both biobrick design and efficiency optimization model. |   Through the modeling build with substituting parameters from published articles, the growth of <i>E. coli</i> and <i>P. gingivalis</i> under the effect of LL-37 would be inhibited significantly. However, the figure also show that <i>E. coli</i> could still live with LL-37. Simply speaking, we successfully test the feasibility of denteeth and find that we can improve the denteeth with both biobrick design and efficiency optimization model. | ||
| Line 72: | Line 73: | ||
</path> | </path> | ||
</svg>Figure 4. The growth curve of <i>E. coli</i> and <i>P.gingivalis</i> with DenTeeth</div> | </svg>Figure 4. The growth curve of <i>E. coli</i> and <i>P.gingivalis</i> with DenTeeth</div> | ||
| + | </div> | ||
| + | <div class="section"> | ||
| + | <h2 class="topic">Test</h2> | ||
| + | <h2 class="subtopic">Model Validation </h2> | ||
| + | |||
<h2 class="subtopic">Functional Test</h2> | <h2 class="subtopic">Functional Test</h2> | ||
<p id="p2"> | <p id="p2"> | ||
Revision as of 14:43, 19 October 2021
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Research
From the research, we know that periodontal disease was proved to be related to many chronic and cardiovascular diseases. Among the pathogenic organisms of periodontal disease, Porphyromonas gingivalis is the most important bacteria that give rise to this bad condition in dogs. To tackle the problem at its roots, we put effort into finding the efficient way to reach the inhibition of Porphyromonas gingivalis first.
Finally, we chose antimicrobial peptides, LL-37 play the part of bacteria-inhibition. LL-37 not only can inhibit P. gingivalis [1], can also inhibit E. coli itself [2], preventing DenTeeth from overgrowing after entering the canine mouth.
Design
Sterilization is the key to periodontal disease. For the wet lab, we first expect the LL-37 could attain the mission. To observe the expression of DenTeeth’s biobrick, the RFP would be inserted behind the LL37. Because DenTeeth would kill by LL-37 peptide too, we added a composite part in the front of the LL-37 producing sequence, preventing engineered bacteria died before entering the mouth.
As stated before, LL-37 can inhibit E. coli. If the DenTeeth could not exist with LL-37 in a proper proportion, the DenTeeth wouldn't attain the mission of solving the periodontal disease. Therefore, we decided to use the prediction model to predict whether DenTeeth could grow and express under the effect of LL-37. To confirm the feasibility of DenTeeth.
Only when the temperature is up than 37°C, the LuxR would be expressed (Fig.1 A). At this moment, if the concentration of bacteria is high, the amount of AHL would rise, binding with LuxR. The complex of AHL and LuxR will attach to the Plux (Fig.1 B), activating the expression of LL-37.
To see more details click to design page.
For the dry part, by deriving differential equations based on our assumptions and substituting parameters from published articles, we could analyze the biological parameter of DenTeeth and predict the expression of LL-37.
Build
Cloning Result
We successfully build engineering bacteria with LL-37.
Through the modeling build with substituting parameters from published articles, the growth of E. coli and P. gingivalis under the effect of LL-37 would be inhibited significantly. However, the figure also show that E. coli could still live with LL-37. Simply speaking, we successfully test the feasibility of denteeth and find that we can improve the denteeth with both biobrick design and efficiency optimization model.
Test
Model Validation
Functional Test
By taking the method of dish culture and sticking up the filter, we drop the engineering bacteria on the filter in the center of the plate. Throughout the process of comparing the circle of inhibiting bacterium, we can prove that LL-37 expressing by our engineering bacteria can inhibit the growth of bacteria.
To see more details click to result page.
Learn
After understanding the effect of LL-37, we ensure the process of denteeth. Due to knowing the effect of LL-37, we take the quorum sensing system to make the denteeth express the LL-37 appropriately. Furthermore, the efficiency optimization model could optimize the benefit of denteeth.
Reference
- Xue Yang, Li Niu, et al.(2020)”LL-37-Induced Autophagy Contributed to the Elimination of Live Porphyromonas gingivalis Internalized in Keratinocytes”Frontiers in Cellular and Infection Microbiology 10:561761
- Xuelian Huang , Keke Zhang et al. (2017)”Effect of arginine on the growth and biofilm formation of oral bacteria”Archives of Oral Biology 82:256-262