Team:Freiburg/Content/Results/BioBrick

From 2014.igem.org

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<ul><li> Gal4BD fused to a LOV domain
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<ul><li> <a href="http://parts.igem.org/Part:BBa_K1470002">Gal4BD</a> fused to a LOV domain
    <li> used for blue light induced gene expression
    <li> used for blue light induced gene expression
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Revision as of 03:40, 18 October 2014

The AcCELLerator

BioBricks

On this page, you will find our new designed BioBricks and also our plasmids used in the experiments.

Here you can get an Overview of our BioBricks. If you want to see more details especially for certain BioBricks, you can click on the BioBrick definition and explore the registry entries.

  • BBa_K1470000: mCAT-1

    mCat-1 is the receptor we introduced to human cells to be able to infect them using the MuLV. The first of our favorite parts! :)

  • BBa_K1470001: Puromycin N-acetyl-transferase (PAC)

    Puromycin N-acetyl-transferase is an enzyme which we used for selecting stable eucaryotic cell lines. Because of the blocked protein biosynthesis more than 99% of uninfected cells will be dead within two days.

  • BBa_K1470002: Galactose-induced gene 4 (Gal4BD)

    Galactose-induced gene 4 (Gal4BD) is used to investigate gene expression levels.

  • BBa_K1470003: Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE)

    Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) is a regulatory RNA sequence, which promotes viral gene expression.

  • BBa_K1470004: Secreted Alkaline Phosphatase (SEAP)

    Secreted Alkaline Phosphatase (SEAP) is used in cell culture assays to quantify gene expression. The second of our favorite parts! :)

  • BBa_K1470005: engineered PDZ domain (ePDZ)

    engineered PDZ domain (ePDZ) is an important part of the blue light system.

  • BBa_K1470006: The Viral Vector (pMIG)

    The Viral Vector (pMIG) is the plasmid which is used to tranfect Phoenix cells with the gene to be packed into viral particles. It contains two LTRs and a multiple cloning site. The third of our favorite parts! :)

  • BBa_K1470007: Cas9-Nickase

    Cas9-Nickase is a Cas9 without iGEM restrction-sites but full catalytic activity.

    • Plasmids

    mCat-1 Plasmids

    Constitutive Expression

  • p14rz_003: pcDNA_SLC7A1_GFP

    • mCAT-1 fusion protein tagged with a C-terminal GFP (green fluorescent protein)
    • used for fluorescent imaging

  • p14rz_004: pcDNA_SLC7A1_HA

    • mCAT-1 fusion protein tagged with a C-terminal HA (hemagglutinin)
    • used for Western Blot studies

  • p14rz_005: pcDNA_SLC7A1_mCherry

    • mCAT-1 fusion protein tagged with a C-terminal mCherry fluorescent protein
    • used for fluorescent imaging

  • p14rz_006: pcDNA_SLC7A1_HA_p2a_mCherry

    • mCAT-1 tagged with a C-terminal HA and mCherry
    • the HA and the mCherry are separated by a P2A sequence that results in 2 separate proteins, mCAT-1-HA and mCherry

  • p14rz_007: pcDNA_SLC7A1_HA_mCherry

    • mCAT-1 tagged with a C-terminal HA and mCherry
    • the HA and the mCherry are linked by a GGSGGSGGSGGSGG-linker resulting in a doubled tagged mCAT-1 fusion protein

    • Red Light induced Gene Expression

  • p14rz_010: TetO13_CMVmin_SLC7A1_HA_p2a_mCherry

    • inducible vector encoding mCAT-1 fusion protein under control of a modified Tet promoter (Ptet) harboring a spacer between the 13mer tetO operator and the minimal promoter (TetO13–CMVmin)
    • the HA and the mCherry are separated by a P2A sequence that results in 2 separate proteins, mCAT-1-HA and mCherry

  • p14rz_011: TetO13_CMVmin_SLC7A1_HA_mCherry

    • inducible vector encoding mCAT-1 fusion protein under control of a modified Tet promoter (Ptet) harboring a spacer between the 13mer tetO operator and the minimal promoter (TetO13–CMVmin)
    • the HA and the mCherry are linked by a GGSGGSGGSGGSGG-linker resulting in a doubled tagged mCAT-1 fusion protein

    • Blue Light induced Gene Expression

  • p14rz_008: Gal4_SLC7A1

    • inducible expression vector encoding mCAT-1 under control of a galactose-inducible promoter (Gal4UAS)
    • in the dark the expression of mCAT-1 gene is off
    • to induce expression of the mCAT-1 gene the pKM292 + pKM297 plasmids are needed

  • p14rz_009: Gal4_SLC7A1_HA

    • inducible expression vector encoding the mCAT-1 fusion protein (tagged with a C-terminal HA) under control of a galactose-inducible promoter (Gal4UAS)
    • in the dark the expression of mCAT-1 gene is off
    • to induce expression of the mCAT-1 gene the pKM292 + pKM297 plasmids are needed

    • Light System

    Blue Light

  • p14ls_002: Gal4_mCherry

    • inducible expression vector encoding the reporter gene mCherry under control of a galactose-inducible promoter (Gal4UAS)
    • in the dark the expression of the reporter gene is off
    • to induce expression of mCherry the pKM292 + pKM297 plasmids are needed

    • Viral Vector Plasmid

    image/svg+xml amp R pUC ori 3´LTR MCS psi_plus_pack 5´LTR PstI (1,463) NotI (1,435) EcoRI (1,426), 2 0 0 4 0 0 6 0 0 8 0 0 1 , 0 0 0 1 , 2 0 0 1 , 4 0 0 1 , 6 0 0 1 , 8 0 0 2 , 0 0 0 2 , 2 0 0 2 , 4 0 0 2 , 6 0 0 2 , 8 0 0 3 , 0 0 0 3 , 2 0 0 3 , 4 0 0 3 , 6 0 0 3 , 8 0 0 4 , 0 0 0 4 , 2 0 0 4 , 4 0 0 4 , 6 0 0 4 , 8 0 0 4 , 9 4 0 BBa_K1470006 (pMIG) 4,940 bp

  • retroviral expression vector with IRES-GFP cassette

  • the vector allows cloning of genes of interest within the iGEM cloning sites EcoRI-NotI-PstI
  • BBa_K1470006
  • one of our favorite parts!

    • Foreign Plasmids

    (many thanks to Konrad Müller, AG Weber)

    Blue Light System

  • pKM292

    • Gal4BD fused to a LOV domain
    • used for blue light induced gene expression

  • pKM297

    • ePDZ fused to the activater domain VP16
    • used for blue light induced gene expression

    • Retrieved from "http://2014.igem.org/Team:Freiburg/Content/Results/BioBrick"