Revision as of 14:46, 21 October 2021

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Overview
Gene Construct
Protein Expression
Functional Test
Feeding Frequency Verification
Safety

Overview

To incorporate quorum sensing part, inhibition part, and restoration part into E. coli, we first built up the gene construct and expressed red and green fluorescent proteins to know our biobricks can be successfully expressed, and test their functions to prove the proteins secreted by DenTeeth can work. Finally, we designed an experiment to ensure the biosafety of DenTeeth.

Gene Construct

We incorporate quorum sensing part, inhibition part, and restoration part into E. coli BL21(DE3). We did colony PCR and digest to check its genotype.

Figure 1. Cloning result after cloning quorum sensing part, inhibition part, and restoration part into E. coli BL21(DE3). (A) Quorum sensing sequence. M : 1kb DNA ladder, 1 : Constitutive promoter + RBS + LuxR + Term. +Term. (1308 b.p.) (B) Quorum sensing sequence + Restoration sequence. M :1kb DNA ladder, 1 : Constitutive promoter + RBS + LuxR + Term. +Term. + Ptet + RBS + BMP2 + RBS + STATH + RBS + GFP + Term. + Term. (2898 b.p.) (C) 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.)

Protein Expression

Functional Test

LL-37 Functional Test

Figure 2. 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 ) )

Figure 3. The Bar Chart of DenTeeth inhibition Functional Test

STATH Functional Test

After making STATH successfully translocated onto the inner membrane of our DenTeeth, we want to know whether the function of STATH works in our DenTeeth.

STATH can prevent the precipitation of calcium phosphate in saliva and maintain a high calcium level in saliva. Therefore, STATH is available for promoting the remineralization of tooth enamel and preventing calculus formation

Calcium Attachment Test

To test whether the STATH expressed by DenTeeth had the function, we decided to detect the calcium ions concentration change with the STATH property of binding to the surface of hydroxyapatite (HA), preventing the calcium ion from attaching to HA. Due to HA being the main component of enamel in teeth, we want to use HA to simulate the oral condition. However, the calcium ion concentration change was too small to detect, and no vendor could provide us HA in time, resulting from the impact of Covid-19.

After the consultation with prof. Hsiao-Ching, Lee, we came up with a replacement plan, observing the attachment of calcium ions on the carbon rod surface. 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.

Feeding Frequency Verification

Safety

To prove that DenTeeth can be inhibited by gastric acid, we conducted an Inhibition of Growth of DenTeeth Test. Because the HCl concentration of the animal's gastric acid ranged from 0.15M to 0.05M, we added 200μL HCl_(aq) from 0.3M to 0.1M with equal DenTeeth and observed whether the DenTeeth in different concentrations of HCl_(aq) would be inhibited. In addition, we chose ddH₂O and HClO_(aq) as our negative control and positive control, respectively.

Figure 1. Result of inhibition of growth of DenTeeth experiment. ( The DenTeeth in plate was in following condition. A. ddH₂O, B. 6% HClO_(aq), C. 0.3M HCl_(aq), D. 0.25M HCl_(aq), E. 0.20M HCl_(aq), F. 0.15M HCl_(aq), G. 0.1M HCl_(aq).)

The DenTeeth's biobrick could successfully degrade in any range from 0.1M to 0.3M HCl_(aq). Only the sample added with ddH₂O has the band in 4685 b.p., which was the length of our biobrick. The cloning result proved that the biobrick could not have the chance to transfer to another bacteria.

Figure 2. Cloning result of inhibition of growth of DenTeeth test( The samples was in following condition. M. 1kb DNA ladder, 1. ddH₂O, 2. 6% HClO_(aq), 3. 0.3M HCl_(aq), 4. 0.25M HCl_(aq), 5. 0.20M HCl_(aq), 6. 0.15M HCl_(aq), 7. 0.1M HCl_(aq).)

Reference

Schink, S. J., et al. (2019). "Death rate of E. coli during starvation is set by maintenance cost and biomass recycling." 9(1): 64-73. e63.

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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">
                      </path>
-                 </svg>Figure 2. 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 : <i> E. coli</i> (BL21) with pSB1K3, C : DenTeeth, D :  <i> E. coli</i> (BL21) with pSB1K3 (Cell Lysate), E : DenTeeth (Cell Lysate), F : PBS, G :  <i> E. coli</i> (BL21) with pSB1K3 + PBS, H : DenTeeth + PBS,  I : LB broth (Centrifuging <i> E. coli</i> (BL21) with pSB1K3 after liquid culturing ), J :  LB broth (Centrifuging <i> E. coli</i> (BL21) with pSB1K3 after liquid culturing ) ). </i></div>
+                 </svg>Figure 2. 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 : <i> E. coli</i> (BL21) with pSB1K3, C : DenTeeth, D :  <i> E. coli</i> (BL21) with pSB1K3 (Cell Lysate), E : DenTeeth (Cell Lysate), F : PBS, G :  <i> E. coli</i> (BL21) with pSB1K3 + PBS, H : DenTeeth + PBS,  I : LB broth (Centrifuging <i> E. coli</i> (BL21) with pSB1K3 after liquid culturing ), J :  LB broth (Centrifuging <i> E. coli</i> (BL21) with pSB1K3 after liquid culturing ) ) </i></div>
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                  <div class="explanation" style="text-align: left;"><svg class="icon" aria-hidden="true" data-prefix="fas" data-icon="arrow-circle-up"
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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">
                      </path>
-                 </svg>Figure ?. </i></div>
+                 </svg>Figure 3. The Bar Chart of DenTeeth inhibition Functional Test</i></div>
                  <h2 class="subtopic" id="topic5">STATH Functional Test</h2>
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