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Revision as of 01:13, 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

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

- 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
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pKM297

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zweites
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@@ Line 55: / Line 55: @@
 <section>
 <h3>Constitutive Expression</h3>
-<h4>p14rz_003: pcDNA_SLC7A1_GFP</h4>
+<li><h4>p14rz_003: pcDNA_SLC7A1_GFP</h4>
 	<div class="row category-row">
 		<div class="col-sm-6">
@@ Line 69: / Line 69: @@
 		</div>
 	</div>
-<h4>p14rz_004: pcDNA_SLC7A1_HA</h4>
+<li><h4>p14rz_004: pcDNA_SLC7A1_HA</h4>
 	<div class="row category-row">
 		<div class="col-sm-6">
@@ Line 83: / Line 83: @@
 		</div>
 	</div>
-<h4>p14rz_005: pcDNA_SLC7A1_mCherry</h4>
+<li><h4>p14rz_005: pcDNA_SLC7A1_mCherry</h4>
 	<div class="row category-row">
 		<div class="col-sm-6">
@@ Line 97: / Line 97: @@
 		</div>
 	</div>
-<h4>p14rz_006: pcDNA_SLC7A1_HA_p2a_mCherry</h4>
+<li><h4>p14rz_006: pcDNA_SLC7A1_HA_p2a_mCherry</h4>
 	<div class="row category-row">
 		<div class="col-sm-6">
@@ Line 111: / Line 111: @@
 		</div>
 	</div>
-<h4>p14rz_007: pcDNA_SLC7A1_HA_mCherry</h4>
+<li><h4>p14rz_007: pcDNA_SLC7A1_HA_mCherry</h4>
 	<div class="row category-row">
 		<div class="col-sm-6">
@@ Line 128: / Line 128: @@
 <section>
 <h3>Red Light induced Gene Expression</h3>
-<h4>p14rz_010: TetO13_CMVmin_SLC7A1_HA_p2a_mCherry</h4>
+<li><h4>p14rz_010: TetO13_CMVmin_SLC7A1_HA_p2a_mCherry</h4>
 	<div class="row category-row">
 		<div class="col-sm-6">
@@ Line 142: / Line 142: @@
 		</div>
 	</div>
-<h4>p14rz_011: TetO13_CMVmin_SLC7A1_HA_mCherry</h4>
+<li><h4>p14rz_011: TetO13_CMVmin_SLC7A1_HA_mCherry</h4>
 	<div class="row category-row">
 		<div class="col-sm-6">
@@ Line 159: / Line 159: @@
 <section>
 <h3>Blue Light induced Gene Expression</h3>
-<h4>p14rz_008: Gal4_SLC7A1</h4>
+<li><h4>p14rz_008: Gal4_SLC7A1</h4>
 	<div class="row category-row">
 		<div class="col-sm-6">
@@ Line 174: / Line 174: @@
 		</div>
 	</div>
-<h4>p14rz_009: Gal4_SLC7A1_HA</h4>
+<li><h4>p14rz_009: Gal4_SLC7A1_HA</h4>
 	<div class="row category-row">
 		<div class="col-sm-6">
@@ Line 195: / Line 195: @@
 <h3>Blue Light</h3>
-<h4>p14ls_002: Gal4_mCherry</h4>
+<li><h4>p14ls_002: Gal4_mCherry</h4>
 	<div class="row category-row">
 		<div class="col-sm-6">
@@ Line 240: / Line 240: @@
 <h2 id="Plasmid-Foreign">Foreign Plasmids</h2>
 <h3> Blue Light System </h3>
-<h4>pKM292</h4>
+<li><h4>pKM292</h4>
 	<div class="row category-row">
 		<div class="col-sm-6">
@@ Line 256: / Line 256: @@
 	        </div>
 	</div>
-<h4>pKM297</h4>
+<li><h4>pKM297</h4>
 	<div class="row category-row">
 		<div class="col-sm-6">

Team:Freiburg/Content/Results/BioBrick