Revision as of 23:53, 21 October 2021

p(tetR) promoter + RBS + BMP2 + RBS + STATH + RBS + GFP + Terminator*2

p(tetR) promoter + RBS + BMP2 + RBS + STATH + RBS + GFP + Terminator*2

Restoration sequence in DenTeeth

   When the number of bacteria decreases, it will activate BMP2 and STATH to repair gums and help tooth remineralization. This mechanism can avoid the formation of biofilm during the remineralization process, and it also can achieve the best efficient restoration on the condition of fewer bacteria.

Figure 1. Restoration sequence

Gene Construct of DenTeeth

  We incorporate the whole biobrick 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>

GFP Model

   Then, we fit the data of GFP. We found that the expression of GFP exceeded expectations. So, we raised the translation rate of GFP and lowered the degradation rate. Although we did the same experiment for 24 hours, since the GFP expression had exceeded the detection range of the machine, the measured values were maintained at the maximum. Therefore, we only took the first 10 hours for validation. The result is shown below.

Figure 3. The fitting result of the intensity of GFP.

STATH and BMP2 Model

   We predicted the BMP2 and STATH expression again.

Figure 3. The prediction of BMP2 and STATH.

STATH Functional Test

   Carbon rods are processed to have small, low-volume pores that increase the surface area available for adsorption. We expected that both STATH and calcium could adsorb on the surface of carbon rods, so we designed a test to observe the surface of carbon rods soaked in different solutions under the microscope.

   We made four solutions configured to calcium ions saturated solutions by the calcium chloride solution, ddH₂O, LB broth, DenTeeth, and E. coli with pSB1K3, in the tubes with the carbon rods stuck inside. After putting them into a 0°C refrigerator for 12 hours, the solutions would become calcium ions supersaturated solutions. In the DenTeeth, STATH would bind onto the surface of the carbon rod, preventing the calcium ions from binding onto it.

   As for the precipitation on the bottom of tubes was the debris of bacteria, which was died of the broken cell membrane caused by the high concentration of calcium chloride solution.

Figure 2. The calcium ions saturated solutions.( (A) ddH₂O, (B) LB broth, (C) DenTeeth, (D) E. coli with pSB1K3)

  The result in the following figure showed that the surface of the carbon rod taken out of the DenTeeth was matte compared with that in other solutions. We presumed that the result was due to the protective layer on the surface formed by the STATH that would prevent the liquid from attachment.

Figure 3. The carbon rods took out from calcium ions saturated solutions.( (A1) ddH₂O, (A2) Original ( did not soak in any solution), (B1) LB broth, (B2) DenTeeth, (B3) E. coli with pET30A)

   We can see the different conditions of calcium attachment on carbon rods after soaking in different calcium ions saturated solutions overnight in Figure 8.   Picture A in figure 8 is the original carbon rod. We can observe many layer-like structures on its surface clearly. Since the original carbon rod hadn't soaked in any solution, no crystal of calcium can be seen on its surface. However, without soaking in any solution, there is some little dust smaller than crystal particles on its surface.

   Picture B is the original carbon rod soaked in calcium ions saturated ddH₂O. Obvious white particles can be observed on its surface. We presume the particles might be the crystal of calcium.

   Picture C is the carbon rod soaked in calcium ions saturated LB Broth. There is a relatively big particle on the surface, in our opinion, is formed by calcium crystals.

   The carbon rod soaked in calcium ions saturated E. coli DenTeeth is shown in Picture D. With STATH functioning, there are no particles be seen on the surface of the carbon rod and the surface remains clear. Besides, the surface of it is even cleaner than the original carbon rod. We suppose that is because this rod has been soaked in the solution, which will bring away the dust on its surface.

   Carbon rod soaked in calcium ions saturated E. coli BL21(DE3) with pET32a. We chose this colony as one of our control groups since it has the same Kanamycin resistance as DenTeeth. There are many particles on its surface, which might be composed of calcium crystals.

Figure 4. The comparison of calcium attachment on carbon rods after soaking in different calcium ions saturated solutions overnight. (A) The original carbon rod. (B) Carbon rod soaked in calcium ions saturated ddH₂O. (C) Carbon rod soaked in calcium ions saturated LB Broth. (D) Carbon rod soaked in calcium ions saturated E. coli DenTeeth. (E) Carbon rod soaked in calcium ions saturated E. coli BL21(DE3) with pET32a.

Sequence and Features