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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
Protein Expression
Functional Test
LL-37 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.
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 colony PCR result proved that the biobrick could not have the chance to transfer to another bacteria.
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.