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− | </svg>Figure 5. LL-37 | + | </svg>Figure 5. LL-37 inhibition Zone</div> |
<img class="images" style=" width: 113%;margin-left: -6.5%;" src="https://static.igem.org/mediawiki/2021/0/03/T--NCTU_Formosa--LL-37_functional_test.png" /> | <img class="images" style=" width: 113%;margin-left: -6.5%;" src="https://static.igem.org/mediawiki/2021/0/03/T--NCTU_Formosa--LL-37_functional_test.png" /> | ||
<div class="explanation" style="text-align: left;"><svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" | <div class="explanation" style="text-align: left;"><svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up" |
Revision as of 13:30, 21 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 (P. 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. Human cathelicidin-derived LL-37 is a 37-residue cationic, amphipathic α-helical peptide. It is an active component of mammalian innate immunity. LL-37 not only can inhibit P. gingivalis [1], can also inhibit Escherichia coli itself [2], preventing DenTeeth from overgrowing after entering the canine mouth.
Image
The study showed that [3], producing the LL-37 peptide by E. coli, would make a change in cell shapes and can permeabilize both cell membranes of E.coli. With scanning electron microscopy, the changes in cell changes observed on E. coli cells were evident. Therefore, we imaged that LL-37 can secrete out of the E. coli without cell lysate.
Design
Inhibition is the key to periodontal disease. In 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 be killed by LL-37 peptide, 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
Colony PCR Result
We successfully built DenTeeth with LL-37 .
Model
Through the modeling built 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.
Image
Because P. gingivalis was in the RG2, so we could not do the LL-37 functional test by inhibit the growth of P. gingivalis. After reading some related papers, we found that the killing rate of E. coli was similar to that of P. gingivalis [4]. Based on these data, we determined to make E. coli, DH5α with pET32A, as the bacteria killed by DenTeeth in the LL-37 functional test.
Parameters | Description | Values | Units |
---|---|---|---|
kk | killing rate [4] | 0.04 | 1/μM·min |
N | LL-37 absorbed per dead cell [4] | 0.35 | μM/O.D |
Test
Model Validation
Functional Test
After finishing the design of DenTeeth, we wanted to know whether its inhibition ability can have a function , so we designed the following experiment.
To see more details click to result page.
Learn
After the engineering of LL-37, we could guarantee LL-37 can secrete out from DenTeeth without cell lysis and inhibit the growth of P. gingivalis. We ensured the process of DenTeeth could work smoothly and 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
- Ján Krahulec, Marcela Hyrsová et al. (2010)”High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli”Appl Microbiol Biotechnol 88(1):167-75
- Kriebel K, Biedermann A, Kreikemeyer B, Lang H , et al(2013). “Anaerobic Co-Culture of Mesenchymal Stem Cells and Anaerobic Pathogens - A New In Vitro Model System.” PLoS ONE 8(11): e78226. doi:10.1371/journal.pone.0078226