Revision as of 22:54, 21 October 2021

Plux promoter + RBS + LL37 + RBS + mRFP + RBS + Terminator*2

Plux promoter + RBS + LL-37 + RBS + mRFP + RBS + Terminator*2

Sterilization sequence in DenTeeth

   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.

Figure 1. Sterilization sequence.

Gene Construct of DenTeeth

  We incorporate the whole part into E. coli BL21(DE3). We did colony PCR and digest to check its genotype.

Figure 2. Quorum sensing sequence + Restoration sequence + Sterilization sequence. M :1kb DNA ladder, 1 : Constitutive promoter + RBS + LuxR + Term. +Term. + Ptet + RBS + BMP2 + RBS + STATH + RBS + GFP + Term. + Term. + Plux + RBS + LL-37 + RBS + mRFP + RBS + tetR + Term. + Term. (4685 b.p.)</i>

mRFP Model

   We inserted mRFP into our biobrick to detect the expression of the sterilization sequence in DenTeeth. The following graph was the simulated expression curve of mRFP.

Figure 3. The simulated expression curve of mRFP

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 shows 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.

Figure 4. The growth curve of E. coli and P. gingivalis

Figure 5. The growth curve of E. coli and P. gingivalis

   Because P. gingivalis was in the RG2, so we could not do the LL-37 functional test by inhibiting 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.

Table 1. Parameters of the sterilization system of LL37

LL-37 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.
   By taking the method of dish culture and sticking up the filter paper, we dropped the DenTeeth on the filter paper in the center of the E. coli, DH5α with pET32a, plate. After twelve hours, a circular area around the spot of the DenTeeth formed, in which the bacteria colonies did not grow. Inhibition zone proved that the LL-37 produced by DenTeeth could successfully secrete out and inhibit the growth of E. coli, DH5α with pET32a.

Figure 6. DenTeeth inhibition zone result in LB plates. ( The materials added in plate were in the following condition, and each total volume is 20μL. (A) 30% hydrogen peroxide, (B) E. coli (BL21) with pSB1K3, (C) DenTeeth, (D) E. coli (BL21) with pSB1K3 (Cell Lysate), (E) DenTeeth (Cell Lysate), (F) PBS, (G) E. coli (BL21) with pSB1K3 + PBS, (H) DenTeeth + PBS, (I) LB broth (Centrifuging E. coli (BL21) with pSB1K3 after liquid culturing ), (J) LB broth (Centrifuging E. coli (BL21) with pSB1K3 after liquid culturing ) )

   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 but can also inhibit Escherichia coli itself, preventing DenTeeth from overgrowing after entering the canine mouth.

Figure 5. The Bar Chart of DenTeeth inhibition Functional Test.

Sequence and Features