Team:HSiTAIWAN/Parts

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　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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  • Parts

    • Framework
    • Basic Part
    • Composite Part
    • Part Collection
    • Reference
    • 　　 　　

    Parts

    Framework

    This year, in our project, we tried to detect copper, lead, arsenic and aflatoxin in Chinese herb medicine. Most of these toxins are included in the list that our government has identified as toxic substances in Chinese Medicine to be monitored. We have submitted four new basic/single parts(BBa_K1961000, BBa_K1961001, BBa_K1961002, BBa_K1961003) and two new composite parts(BBa K1961008, BBa_K1961009) for generation RecF(BBa_K1961001) and RecO(BBa_K1961002), respectively. These new parts are all for the project of biosensor for aflatoxin.

    Among the four parts of function improvement(BBa_K1961004, BBa_K1961005, BBa_K1961006, BBa_K1961007), we designed two new circuits of I721002 for measuring PbrR generation (BBa_K1961004, BBa_K1961005). These two new composite parts, BBa_K1961004 and BBa_K1961005, are for modeling purposes in order to have better measurement of PbrR generation measured by GFP expression. We designed an auto-regualted PbrR generator(BBa_K1961004) and a general PbrR generator(BBa_K1961005). We re-ligased or submitted parts that were the previously registered but unsubmitted parts (BBa_K1961006 for BBa_K1613017 and BBa_K1961007 for BBa_1613004).

    Diagram 1. Total Parts in The Herb Tasters, 2016 iGEM.

    SP, single part(=basic part); CP, composite part

    Basic Part

    We have 4 new basic parts for aflatoxin biosensors and we have also submitted one part of function improvement of K1613017(previously unsubmitted part)

    For the biosensor of aflatoxin, we submitted four basic parts. Detection of aflatoxin is involved of two steps: (1) aflatoxin metabolization, which creates single-stranded DNA and activated Rec A; and (2) SOS induction: activated RecA cleaves the LexA repressor on SOS response.

    For the aflatoxin metabolism step, we know that among the Cytochrome P450 enzypmes, CYP3A4(K1064004)/CYP3A5 and CYP1A2(K1613004) are major liver enzymes that metabolize aflatoxin. We reviewed literature and found CYP2A13(BBa_K1961000), which is a Cytochrome P450 in human lung and metabolizes aflatoxin, too. This new single part can be included in the experiment of aflatoxin biosensor.

    For the SOS induction step, we found in literature that RecF (BBa_K1961001), RecO(BBa_K1961002),and RecR(BBa_K1961003) proteins in RecFOR pathway mediate the loading of RecA protein onto ssDNA 1. RecA polymerizes on single-stranded (ss) DNA and form nucleoprotein filaments. The filament induces LexA repressors cleavage and result in SOS induction. Thus, RecF, RecO, and RecR are important “mediator”proteins that determine SOS induction. Thus, we submitted them as new parts.

    Part Type Description
    BBa_K1961000 coding cytochrome P450 family 2 subfamily A member 13 (CYP2A13)
    BBa_K1961001 coding RecF protein-SOS mediator protein. It is involved in the RecFOR pathway for DNA metabolism; it is required for DNA replication and normal SOS inducibility.
    BBa_K1961002 coding RecO protein-SOS mediator protein. It is involved in the RecFOR pathway for DNA metabolism; it is required for DNA replication and normal SOS inducibility.
    BBa_K1961003 coding RecR protein-SOS mediator protein. It is involved in the RecFOR pathway for DNA metabolism; it is required for DNA replication and normal SOS inducibility.
    BBa_K1961007 coding CYP1A2 aflatoxin metabolism
    A single part that contain CYP1A2 code.
    　　

    Composite Part

    We have two new composite parts for the Pb2+ biosensor for Chinese herbal medicine, and a part for function improvement for BBa_K1613017.

    1. The Pb2+ biosensor (BBa_K1961006):it is a function improvement for BBa_K1613017 (HSNU-Tapei, 2015 iGEM). We re-ligased this composite part. This composite part involves two stages (1) generation of PbrR promoter with a binding site for lead ion, thus forming Pb2+-PbrR dimer and (2) fluorescence expression when Pb2+-PbrR dimer binds onto pbr promoter (Figure 1) or when PbrR binds onto pbr promoter first followed by lead ion binding onto it (Figure 2)

    Figure 1 Lead ion detector: dimer binds onto pbr promoter

    Figure 2 Lead ion detector: PbrR and lead ion bind onto pbr promoter sequentially

    From literature, we understand that the PbrR and Pb2+-PbrR dimer compete to bind onto pbr promoters and result in poor fluorescence performance. In order to measure the PbrR generation, we designed an autoregulated PbrR generator (BBa_K1961004 ) to control the amount of PbrR and compared its performance with a common PbrR generator (BBa_K1961005 ).

    2. Autoregulated PbrR generator(BBa_K1961004): consists of the TetR and PbrR generators, both controlled by PTet, which has TetR binding sites. TetRs bind onto PTet and repress the gene expression of TetR, PbrR and GFP. We used tetracycline to induce the gene expression of PbrR and GFP (Figure 3)

    Figure 3. Autoregulation PbrR generator circuit: the generation of PbrR measured by GFP expression downstream and is under the control of TetR and promoter PTet

    3. Common PbrR generator (BBa_K1961005 ): the generation of PbrR is under control of a constitutive promoter (BBa_J23102 ) (Figure 4)

    Figure 4. Constitutive PbrR is used to measure the background expression of PbrR in E. Coli without any inducer

    The performance of the autoregulated generator of PbrR was compared with the generator of PbrR with a general promoter .

    Part Type Description
    BBa_K1961004 generator PbrR generator expressed by GFP under autoregulation of PTet, which is regulated by TetR repressor. Meanwhile, the transcription of TetR is also under control of PTet promoter. R0040+B0034+C0040+B0010+B0012+R0040++B0032+I721002+E0040+B0010+B0012
    BBa_K1961005 generator PbrR generator expressed by GFP under control of general promoter (J23102). J23102+B0032+I721002+E0040+B0010+B0012
    BBa_K1961006 reporter Lead biosensor expressed by RFP expression J23102+B0032+I721002+B0010+B0012+I721001+B0034+E1010+B0010+B0012

    On the other hand, we ligased two new composite parts for the RecF (BBa_K1961001) and RecO(BBa_K1961002) proteins in RecFOR pathway mediate the loading of RecA protein onto ssDNA.

    Part Type Description
    BBa_K1961008 generator Generator of RecF protein-SOS mediator protein
    To enhance DNA replication and repair RecF protein is involved in the RecFOR pathway for DNA metabolism; it is required for DNA replication and normal SOS inducibility.
    BBa_K1961009 generator Generator of RecO protein-SOS mediator protein
    To enhance DNA replication and repair RecO protein is involved in the RecFOR pathway for DNA metabolism; it is required for DNA replication and normal SOS inducibility.
    　　

    Part Collection

    We designed four biosensors to detect Cu2+, As3+, Pb2+, and aflatoxin in the Chinese herb medicine. We have new part collections for Pb2+ and aflatoxin biosensors.

    For the biosensor of Pb2+, literature indicates that the PbrR homodimer and Pb2+-PbrR dimer compete for the binding sites on pbr promoters, resulting in poor performance of report gene (GFP). Thus, we designed an autoregulated -circuit for PbrR generator (BBa_K1961004) to control and measure PbrR. We compared the autoregulated PbrR generator with another PbrR generator with general promoter (BBa_K1961005). Our experiment provides helpful information for future design of new part.

    For the biosensor of aflatoxin, we identified CYP2A13 (BBa_K1961000), a cytochrome P450 in human lung that metabolizes aflatoxin. We also understood that RecF (BBa_K1961001), RecO (BBa_K1961002), and RecR (BBa_K1961003) proteins mediate the loading of RecA protein onto single-stranded DNA, and are important for the SOS induction. Thus, we submitted them as new parts.

    Biosensor of lead ion

    To detect lead ions in Chinese medicine herb, we need a lead-specific binding protein (PbrR), a MerR family of proteins, which is involved in Pb2+ resistance. Like any other MerR family, PbrR proteins are metal-binding and DNA-binding proteins. In the absence of Pb2+, PbrR binds as a homodimer to the divergenet promoter region of PbrR gene and lead-resistence pbr operon, repressing the transcription of PbrR and genes in pbr operon.(Figure 5).

    Figure 5 Lead ion detector: PbrR and lead ion bind onto pbr promoter sequentially

    When there exist Pb2+ in cytoplasm, Pb2+ and PbrR form a Pb2+-PbrR dimer which leads to an allosteric underwinding of the pbr promoter DNA thereby shortening the space between RBS and promoter and activate the transcription.(Figure 6) .

    Figure 6 Lead ion detector: dimer binds onto pbr promoter

    In this sense, both PbrR homodimer and Pb2+-PbrR dimer compete for the binding sites for pbr promoters. Therefore, we introduced a new part-design for autoregulating the transcription of PbrR using the TetR respressor. We used the GFP expression downstream to measure the PbrR(BBa_K1961004) (Figure 7(a)) and compared it with another PbrR generator under control of a constitutive promoter(BBa_K1961005) (Figure 7(b))

    Figure 7(a). The autoregulated PbrR circuit Figure 7(b). The constitutive PbrR circuit

    Parts we submitted

    Part Type Description
    BBa_K1961004 generator Autoregulated PbrR generator
    PbrR generator expressed by GFP under autoregulation of PTet, which is regulated by TetR repressor. Meanwhile, the transcription of TetR is also under control of itself R0040+B0034+C0040+B0010+B0012+R0040++B0032+I721002+E0040+B0010+B0012
    BBa_K1961005 generator common PbrR generator
    PbrR generator expressed by GFP under control of general promoter J23102+B0032+I721002+E0040+B0010+B0012
    BBa_K1961006 reporter Lead biosensor expressed by RFP expression
    J23102+B0032+I721002+B0010+B0012+I721001+B0034+E1010+B0010+B0012

    Biosensor of aflatoxin

    Detection of aflatoxin in Chinese herb medicine is involved with two steps: one is aflatoxin metabolism (Figure 8) and the other one is the SOS response (Figure 9).

    In the first step, we submitted four basic parts: CYP2A13(BBa_K1961000) , RecF(BBa_K1961001), RecO(BBa_K1961002), and Rec(BBa_K1961003). We also used a composite part CYP1A2 (which was designed by HSNU 2015) to metabolize aflatoxin, creating aflatoxin B1 exo-8,9-epoxide(AFBO). AFBO caused DNA damage, creating single-stranded DNA. Single-stranded DNA activates RecA protein, which will make the LexA repressor cleave and proceed the SOS response. RecF, RecO, and RecR proteins mediate the loading of RecA protein onto ssDNA,functioning as the RecOR or RecFOR complexes;these proteins are often called “mediator” proteins.(Figure 8).

    In the second step, we used the part of DNA damage reporter (BBa_K079050, designed by IGEM08 Bologna), which has a SOS box with LexA binding site.(Figure 9).

    Parts we submitted

    Part Type Description
    BBa_K1961000 coding Cytochrome P450, family 2, subfamily A, polypeptide 13(CYP2A13)
    To metablize aflatoxin and related mycotoxin. Like CYP3A4, CYP2A13 is a member of the cytochrome P450 proteins, which oxidizes a variety of structurally unrelated compounds, including steroids, fatty acids, and xenobiotics. CYP3A4, CYP3A5 and CYP1A2 are mainly in liver. CYP2A13 is in lung.
    BBa_K1961001 coding RecF protein-SOS mediator protein
    To enhance DNA replication and repair RecF protein is involved in the RecFOR pathway for DNA metabolism; it is required for DNA replication and normal SOS inducibility.
    BBa_K1961002 coding RecO protein-SOS mediator protein
    To enhance DNA replication and repair RecO protein is involved in the RecFOR pathway for DNA metabolism; it is required for DNA replication and normal SOS inducibility.
    BBa_K1961003 coding RecR protein-SOS mediator protein
    To enhance DNA replication and repair RecR protein is involved in the RecFOR pathway for DNA metabolism; it is required for DNA replication and normal SOS inducibility.
    BBa_K1961008 composite part Generator of RecF protein-SOS mediator protein
    To enhance DNA replication and repair RecF protein is involved in the RecFOR pathway for DNA metabolism; it is required for DNA replication and normal SOS inducibility.
    BBa_K1961009 composite part Generator of RecO protein-SOS mediator protein
    To enhance DNA replication and repair RecO protein is involved in the RecFOR pathway for DNA metabolism; it is required for DNA replication and normal SOS inducibility.

    Parts we improved function

    In addition to the four new parts, we submitted a part(BBa_K1961007) as function improvement for the previously existing but unsubmitted part for CYP1A2(BBa_K1613004)

    Part Type Description
    BBa_K1961007 coding CYP1A2 aflatoxin metabolism
    A single part that contain CYP1A2 code.

    Parts we used

    We used the Aflatoxin detector (BBa_K1613007) to metabolize aflatoxin. We used the SOS part(BBa_K079050, iGEM 2008 Bologna) to test the SOS response induced by actived-Rec A protein, which facilitates the cleavage of LexA transcription repressor on SOS response.

    Part Type Description
    BBa_K079050 device DNA damage reporter
    GFP reporter protein under the control of the J23100 constitutive promoter and LexA 2 operator J23100+K079040+B0034+J04031+B0010+B0012

    Biosensor of arsenic ion

    We used the part of GFP generator under Ars promoter(BBa_K1961004), designed by iGEM13_Buenos_Aires, to detect the arsenic ions in the Chinese medicine. This part consists of a promoter(J33201) with ArsR repressor gene encoding the arsR repressor protein, which is sensitive to sodium arsenate or sodium arsenite. Arsenite or arsenite ion binds to the repressor protein ArsR, resulting in inability to repress the promoter.

    Parts we used

    Part Type Description
    BBa_K1106004 device Arsenic ion detector
    J33201+B0030+E0040+ B0010+B0012

    Biosensor of copper ion

    We used the device of The CopA promoter with a gfp reporter (BBa_K1555000), designed by iGEM14_NJAU_China , to detect copper ions in Chinese medicine. This device includes a sequence which is indirectly regulated by copper through copper-responsive protein CueR and therefore is sensitive to copper ions.

    Parts we used

    Part Type Description
    BBa_K1555000 device Copper ion detector
    Copa promoter+B0030+E0040+B0010+B0012

    Reference

    [1] Katsumi Morimatsu,Yun Wu, StephenC.Kowalczykowski. RecFOR Proteins Target RecA Protein to a DNA Gap with Either DNA or RNA at the5 Terminus. THE JOURNAL OF BIOLOGICAL CHEMISTRY 2012. 287( 42): pp. 35621–35630