Team:HokkaidoU Japan/Notebook/Promoter Construct Promoter Optimization Kit

From 2013.igem.org

Maestro E.coli

Notebook

Construct Promoter Optimization Kit

  • Start

  • PCR & PCR purification

    vector:promoter(K1084001~010)-B0034-mRFP1-B0015-pSB1C3
    V3-F, V1-R, KOD plus Neo / V3-F, V2-R, KOD plus Neo
    Insert:R0040-B0034-lacZα-B0015
    i1-F, i3-R, KOD plus Neo / i2-F, i3-R, KOD plus Neo

  • Digestion

    Cut V1:Promoter-βBsaI-BsaIψ-B0015-pSB1C3 with BsaI
    Cut V2:promoter-B0034-γBsaI-BsaIψ-B0015-pSB1C3 with BsaI
    Cut i1:βBsaI-R0040-B0034-lacZα-B0015-BsaIψ with BsaI
    Cut i2:γBsaI-R0040-B0034-lacZα-B0015-BsaIψ with BsaI

  • Ligation

    Ligate V1 with i1, V1 with i2, V2 with i1, V2 with i2

  • Transformation

    i1 on V1, i2 on V1, i1 on V2, i2 on V2
    5µL DNA to DH5α

  • Colony PCR

    i1 on V1, i2 on V1, i1 on V2, i2 on V2

  • PCR & PCR purification

    pormoter(K1084001~010)-B0034-lacZα-B0015
    100b up, 200b dn, KOD plus Neo

  • Digestion

    Cut B0034-lacZα-B0015-pSB1AK3 with EcoRI, PstI(using 10xH buffer)
    Cut R0040-B0034-lacZα-B0015 with EcoRI, PstI(using 10xH buffer)

  • Ligation

    Ligate R0040-B0034-lacZα-B0015 with pSB1AK3

  • Transformation

    R0040-B0034-lacZα-B0015 on pSB1AK3
    5µL DNA to DH5α

  • PCR & PCR purification

    aeBlue
    gg-XbaI-F, 200b dn, KOD plus Neo

  • Digestion

    Cut R0010 with EcoRI, SpeI
    Cut B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015 with XbaI, PstI
    Cut R0010-B0034-mRFP1-B0015 on pSB1C3 with EcoRI, PstI

  • Ligation

    Ligate R0010 with aeBlue/AmilCP/eforRed/AmilGFP, pSB1C3

  • Transformation

    R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015 on pSB1C3
    5µL DNA to DH5α

  • PCR & PCR purification

    R0040-B0034-lacZα-B0015
    i1-F, i3-R, KOD FX Neo / i2-F, i3-R, KOD FX Neo

  • Digestion

    Cut i1 with BsaI
    Cut i2 with BsaI

  • EtOH precipitation

    i1, i2

  • Digestion

    Cut i1 with BsaI
    Cut i2 with BsaI

  • Ligation

    Ligate V1 with i1, V1 with i2, V2 with i1, V2 with i2

  • Transformation

    i1 on V1, i2 on V1, i1 on V2, i2 on V2
    5µL DNA to DH5α

  • Culture

    i1 on V1, i2 on V1, i1 on V2, i2 on V2

  • PCR

    R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015
    100b up, 200b dn, KOD FX Neo

  • Digestion

    Cut R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015 with XbaI, PstI(using 10xM buffer)
    Cut i1 on V1(i2 on V1/i1 on V2/i2 on V2) with SpeI, PstI(using 10xH buffer)

  • EtOH precipitation

    Digestion product

  • Ligation

    Ligate R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015 with [i1 on V1](i2 on V1/i1 on V2/i2 on V2)

  • Transformation

    R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015-[i1 on V1](i2 on V1/i1 on V2/i2 on V2)

  • Mini-prep

    R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015-[i1 on V1](i2 on V1/i1 on V2/i2 on V2)

  • PCR & Gel extraction

    E-X/S-P on pSB3K3
    Km-gamma-f, Km-psy-r, KOD FX Neo

  • EtOH precipitation

    J31003

  • Digestion

    Cut R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015-[i1 on V1](i2 on V1/i1 on V2/i2 on V2) with BsaI
    Cut J31003 with BsaI

  • EtOH precipitation

    Digestion product

  • Ligation

    Ligate R0010-B0034-aeBlue(AmilCP/eforRed/AmilGFP)-B0015-[i1 on V1](i2 on V1/i1 on V2/i2 on V2) with J31003

  • Transformation

    Ligation product
    5µL DNA to DH5α

  • Culture

    Transformation product
    to LBK plate

  • Complete!

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