Team:HSiTAIWAN/Project

X

　Oh hello there,I am Charlie,a trusty member of the "Herb Tasters" and also the brainiest E. coli in the colony.
　I know all the secrets of Chinese herbs and their magical healing powers.
　If you are up to a challenge,find me at team HSiTW at the jamboree.
　I am the one in a straw hat,showing them pearls.I will be waiting.
　Muhahahahaha

　Hi there! My name is Nu Zhen Chi. This is how I look like.
　　Take a closer look; guess which part of me is used as medicine?
(1) the root
(2) the stem
(3) the leaf
(4) the seed

　Ans.(4) the seed
　Name: 女貞子 (Nu Zhen Chi)
　Botanical Name: Ligustrum lucidum Aiton
　I can treat people who are yin deficient, and liver problems that cause dizziness,cataract of the eyes,
lower back pain, premature graying of the hair and tinnitus.

　Hello! My name is Chuan Xiong. This is how I look like.
　Make a guess, which part of me is used as medicine?
(1) the root
(2) the stem
(3) the leaf
(4) the seed

　Ans.(1) the root
　Name: 川芎 (Chuan Xiong)
　Botanical Name: Ligusticum chuanxiong Hort
　I help with blood regulation to prevent relevant to blood stasis and non-stop bleeding.I can also strengthen your qi circulation.
　In addition, I relieve you of physical pain, such as headaches, abdominal aches, chest pain, and muscle pain.
　Finally, I free the ladies of menstrual disorders and amenorrhea.

　What’s up? My name is Dang Gui. I can:
(1) stop coughing
(2) regulate mense
(3) reduce internal heat

　Ans.(2) Regulate mense
　Name: 當歸 (Dang Gui)
　Botanical Name: Angelica sinensis (Oliv.) Diels
　I can remove blood stasis and clots, so I am usually used to regulate menses,lubricate intestines to correct constipation, reduce swelling, expel pus.

　　Reference
　臧堃堂 (2005) 中華材輕百科-現代版本草綱目，山岳文化出版社，台北
　Non-Profit Organization Brion Research Institute of Taiwan.
　Chinese Herb Gallery. Jade Institute
　Herbal Glossary. Shen-Nong- Chinese Traditional Medicine
　　Acknowledgement
　Thank you for Non-Profit Organization Brion Resaerch Institute of Taiwan that provide us Chinese herbs and photos.

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  • Project of Lead Biosensor

    • Introduction
    • Regulation
    • Circuit Design
    • Reference
    • 　　 　　

    Project of Lead Biosensor

    Introduction

    The brilliant advances in genetic engineering technologies based on proteins and non-coding small RNAs are enabling the systematic biological functions for both prokaryote and eukaryote. In this study, we utilize the mean field theory and the 4th Runge-Kutta method to mimic the engineering synthetic genetic circuits in Escherichia coli (E. coli), including the small RNA (sRNA) regulations and autoregulation circuits. For sRNA level in E. coli, the near simultaneously regulations that decrease (or increase) the target mRNA disable (enable) the target protein function. Therefore, the phase space for induction-regulation plays a key role and should be thoroughly investigated by simulations. In comparison with the sRNA level, the simulations and experimental methods based on the autoregulation circuit (Ptet-tetR) are designed to quantitatively describe the reporter protein or fluorescence response. The Hill coefficient is also checked to disclose the intrinsic properties of the autoregulation circuits in E. coli.

    Regulation

    The introduction for genetic circuits

    Negative regulation —Protein (repressor) inhibits transcription (Ex. LacI, TetR protein). Inducer– binds to repressor, alters form, reduces affinity for target, allows expression of gene. Sometimes, small molecule required for repressor activity.

    Positive regulation —Activator protein increases transcription rate. Generally bound to a smaller signal molecule. (Ex. XylR protein activates Pu promoter).

    sRNA-mediated gene silencing -(sRNA) are small (50-250 nucleotide) non-coding RNA molecules produced by bacteria; they are highly structured and contain several stem-loops. sRNAs can either bind to protein targets, and modify the function of the bound protein, or bind to mRNA targets and regulate gene expression. (Ex. RyhB and sodB)

    Fig 1. Negative regulation: The TetR protein/Tc

    Fig 2. Positive regulation :The XylR protein/Pu promoter

    Fig 3. sRNA-mediated regulation: The RhyB-sodB RNA

    Fig 4. The Constitutive expression: The Pcon-tetR

    Fig 5.The repression-induction switch: The Ptet-tetR

    Fig 6.The sRNA-mediated regulation circuit

    Fig 7.The famous sRNA in E. coli

    Circuit Design of Lead Biosensor

    The introduction for binding constant

    The dissociation constant K

    Fig 8. The cartoon picture illustrating the the dissociation constant K and original of Hill coefficient n where n = 2.

    The Hill coefficient n

    Equation 1: The Hill equation: [R] as the concentration of reporter protein, [I] as the concentration of inducer and KM as the concentration of inducer when [R] reaches to the half of [Rmax]

    Fig 9. The cartoon picture illustrating the the Hill coefficient n

    Modeling method

    The sRNA based genetic circuits

    Fig 10.The complex designed regulation: The RhyB-sodB : w/o sodB

    Fig 11.The complex designed regulation: The RhyB-sodB : w/ sodB

    Fig 12.The sRNA-mediated regulation circuit

    Equation 2: The mean field theory is used to describe the sRNA-mediated regulation circuit.

    Equation 3: The steady state analysis for sRNA-mediated regulation circuit.

    Equation 4: The approximation approach based on the certain condition of transcription rate

    Constitutive circuit

    Fig 13. The Constitutive circuit and the related rate equations

    A regular constitutive circuit contains multiple inducers and repressors with unidirectional regulations.

    Autoregulation circuit

    Fig 14. The Autoregulation circuit and the related rate equations

    Constitutive PbrR circuit

    Fig 15. Constitutive PbrR is used to measure the background expression of PbrR in E. Coli without any inducer

    Autoregulation circuit

    Fig 16. Autoregulation PbrR generator circuit: the generation of PbrR measured by GFP expression downstream and is under the control of TetR and promoter PTet

    Equation 5: The fold analysis of Constitutive Circuit

    Equation 6: The fold analysis of Autoregulation Circuit

    Equation 7: The fold analysis of Autoregulatory and Constitutive circuits When [I] goes to 0, solve numerically.

    Simulation

    The usage of our phase diagram can assist researchers to detect change under solution of different concentration and regulate the concentration of inducer.

    Fig 17. The 2-D phase space for sRNA meediated circuit

    Fig 18. The 3-D phase space for sRNA meediated circuit

    Fig 19. The parameter relationship for circuits

    Fig 20. Fold relationship for Autoregulatory and Constitutive circuits

    Reference

    [1] E. Levine, Z. Zhang, T. Kuhlman, T. Hwa, Quantitative Characteristics of Gene Regulation Mediated by small RNA, PLoS Biol 5 (2007) 1998-2010.

    [2] David Braun et al. Parameter estimation for two synthetic gene networks: A case study. ICASSP (2005) 769-772.

    [3] Timothy S. Gardner Charles R. Cantor & James J. Collins, Construction of a genetic toggle switch in Escherichia coli, Nature 403 (2000) 339-342.

    [4] O. Scholz, P. Schubert, M. Kintrup and W. Hillen, Tet repressor induction without Mg2+, Biochemistry 39 (2000) 10914–10920.

    [5] Peter Orth et al., Conformational changes of the Tet repressor induced by tetracycline trapping, J. Mol. Biol. 279 (1998) 439-447.

    [6] Thomas A Geissmann and Daniele Touati, Hfq, a new chaperoning role: binding tomessenger RNA determines access for small RNA regulator, The EMBO J. 23 (2004) 396–405.

    [7] Nicolae RaduZabet, Negative feedback and physical limits of genes, J. of Theoretical Biology 284 (2011) 82–91.