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

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

LL-37 can promote the autophagy process to eliminate P. gingivalis, the killer of causing periodontal disease, in cells. This way is more efficient than killing the bacteria directly. Also, LL-37 has additional roles such as regulating the inflammatory response in wounds or infection sites and promoting wound closure. Some studies demonstrated that LL-37 has a key role in wound regeneration through vascularization. These can help the oral wound heal faster.

Inhibition 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. Inhibition sequence.

Improvement of LL-37 Performance

LL-37 is an antimicrobial peptide from humans, to improve its performance, we performed codon harmonization. The optimized sequence of codons will be more suitable for expression by our engineered strains than the natural sequence.

Organisms\Peptide	A	C	D	E	F	G	H	I	K	P	R	Y
Homo sapiens	GCC	TGC	GAC	GAG	TTC	GGC	CAC	ATC	AAG	CCC	AGG	TAC
E. coli	GCT	GCG	ATG	AAT	TTG	GCC	ATA	TTA	AAA	CCG	CGC	TAT

Table 1. The comparison of the codon in homo sapiens and in E. coli.

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.

we considered the expression of RFP and fit the model with experimental data as before. We found that the environment of the Erlenmeyer Flask was different from the paper. The degradation of RFP was lower than expected. Thus, we lowered the degradation rate and verified it with the experimental results again. The following picture is the result.

Figure 3. The fitting result of the intensity of RFP

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. 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

Feeding Frequency Validation

Figure 6. A is the control group, and B is the experiment group

In order to validate the efficiency optimization model usable in any environment, we use E. coli with pSB1K3, hydrogen peroxide, and glucose to simulate as P. gingivalis, feeding dental bones and eating foods. As a result of the different environments of the experiment and the dog's mouse. We modified the environment reaction function to meet requirements.

Figure 7. The Validation of Efficiency Optimization Model

The above figure shows that the prediction value is very close to the experimental data, and the prediction is very accurate and precise by observing the R-square and RMSE.

After validating the accuracy and precision of the model, we further compare the reward of two dental bone feeding policies. Policy 1 (control group) is feeding dental bone with a fixed time interval. Policy 2 (experimental group) is feeding dental bone with RL prediction results.

Figure 8. The comparison of Reward from Policy 1 and Policy 2

Through calculating the reward of policy 1 and policy 2 by reward function, we can claim the reward of policy 2 is statistically higher than policy 1. Simply speaking, through the validation experiment, we successfully proved the optimization ability of this model.

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. Our team decided to use the inhibition zone experiment to confirm that DenTeeth can inhibit other bacteria. Furthermore, we can also compare the difference of inhibition zone diameter to know the strength of inhibition intensity.

First, we spread E. coli (DH5α) with pET32a in LB plates with AMP resistance to be our inhibition target. Second, sticking up the filter paper, we dropped the following materials on the filter paper. Finally, we placed LB plates at 37 Celsius degrees for 12 hours.

There are ten different materials added to LB plates. Plate A was added with hydrogen peroxide as the positive control group. We could see that Plate A only grew bacteria near the edge. Plates B and C are E. coli (BL21) with pSB1K3 and DenTeeth, respectively. In these two plates, both of which contained K resistance. With other factors unchanged, Plate B is negative control. As we could see, our DenTeeth had an inhibitory effect. In plate D and plate E, are cell lysate of E. coli (BL21) with pSB1K3 and DenTeeth, respectively. In these two plates, both of which contain K resistance. With other factors unchanged, Plate D is negative control. As we could see, the cell lysate of DenTeeth had an inhibitory effect. According to the results of plate A to plate E, we could know whether it is a live DenTeeth or a cell lysate, both of which had the function of inhibiting bacteria.

In plates F, G and H, are added with PBS, E. coli (BL21) with pSB1K3 + PBS and DenTeeth + PBS, respectively. To ensure PBS would not influence the inhibition results significantly, we decided to make three plates containing PBS because it would be used on cell lysis. In these three plates, we could observe that only plate H (DenTeeth + PBS) had an inhibitory effect. This result allowed us to know that PBS would not have an obvious influence on our experiment, and can know that DenTeeth still had an inhibitory effect.

In plates, I and J, are the LB broth after centrifugation from E. coli (BL21) with pSB1K3 and centrifugation from DenTeeth, respectively. With other factors fixed, we could observe that plate J had an inhibitory effect. This result allowed us to know that antimicrobial peptide LL-37 can be secreted from DenTeeth to the environment.

Figure 9. 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 ) )

We repeated the DenTeeth inhibition functional test three times each. In figure 5, this bar graph indicates the three times average of inhibition zone diameters in different materials added to LB plates. This result shows that DenTeeth can secrete LL-37 to inhibit other bacteria.

Figure 10. The Bar Chart of DenTeeth inhibition Functional Test.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1650
Illegal NgoMIV site found at 1771
Illegal AgeI site found at 778
Illegal AgeI site found at 890
1000
COMPATIBLE WITH RFC[1000]