Difference between revisions of "Team:NCTU Formosa/Results"

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Revision as of 20:42, 20 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

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

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Figure ?.

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 result of PCR cloning is as follows. The DenTeeth could successfully be inhibited in any range from 0.1M to 0.3M 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

  1. 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.
  2. 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
  3. 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
  4. 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
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