Team:Freiburg/Content/Results/BioBrick

From 2014.igem.org

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<h2 id="Plasmid-Light-Systems">Light System</h2>
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<h3>Red Light</h3>
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<h3>Blue Light</h3>
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<h3>Blue Light</h3>
 
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<h4>p14ls_001: COP1-VP16_IRES_TetR-UVR8</h4>
 
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<ul><li>bistronic vector encoding  COP1–VP16 and TetR-UVR8 under control a SV40 promoter
 
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<li>herpes simplex virus (HSV) transactivator protein VP16
 
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<h4>p14ls_002: Gal4_mCherry</h4>
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<h3>Reporter Proteins</h3>
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<h3>Reporter Proteins???</h3>
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Revision as of 00:05, 18 October 2014

The AcCELLerator

BioBricks

On this page, you will find our BioBricks and our used plasmids.

BBa_K1470000: mCAT-1

mCat-1 is the receptor we introduced to human cells to be able to infect them using the MuLV

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.

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.

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 … 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 … 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 … plasmid is/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 … plasmid is/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 … plasmid is/are needed

Viral Vector Plasmids

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

Reporter Proteins???

Foreign Plasmids

Blue Light System

pKM292

  • erstes
  • zweites
  • drittes

pKM297

  • erstes
  • zweites
  • drittes

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