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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 experiment to ensure the biosafety of DenTeeth.

Gene Construct

Figure 1. Colony PCR result of after cloning 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

Figure ?.

Feeding Frequency Verification

Safety

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).)

Figure 2. Colony PCR 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.
Kim C, Wilkins K, Bowers M, Wynn C and Ndegwa E., et al. (2018). "Influence of Ph and Temperature on Growth Characteristics of Leading Foodborne Pathogens in a Laboratory Medium and Select Food Beverages.” Austin Food Sci. 2018; 3(1): 1031
Kriebel K, Biedermann A, Kreikemeyer B, Lang H , et al(2013). “Anaerobic Co-Culture of Mesenchymal Stem Cells and Anaerobic Pathogens - A New In Vitro Model System.” PLoS ONE 8(11): e78226. doi:10.1371/journal.pone.0078226
Snoussi, M., Talledo, J. P., Del Rosario, N. A., Mohammadi, S., Ha, B. Y., Košmrlj, A., & Taheri-Araghi, S., et al (2018). Heterogeneous absorption of antimicrobial peptide LL37 in Escherichia coli cells enhances population survivability. eLife, 7, e38174. https://doi.org/10.7554/eLife.38174

Team:NCTU Formosa/Results