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Revision as of 04:05, 17 October 2014

The AcCELLerator

BioBricks

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

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

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

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

Red Light

Blue Light

UV Light

p14ls_001: COP1-VP16_IRES_TetR-UVR8

bistronic vector encoding COP1–VP16 and TetR-UVR8 under control a SV40 promoter o E3 ubiquitin-protein ligase COP1 o herpes simplex virus (HSV) transactivator protein VP16

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

@@ Line 97: / Line 97: @@
 	<div class="row category-row">
 		<div class="col-sm-6">
-		<ul><li>mCAT-1 tagged with a C-terminal HA and mCherry</p>
+		<ul><li>mCAT-1 tagged with a C-terminal HA and mCherry</li>
-		    <li>the HA and the mCherry are separated by a P2A sequence that results in 2 separate proteins, mCAT-1-HA and mCherry</p>
+		    <li>the HA and the mCherry are separated by a P2A sequence that results in 2 separate proteins, mCAT-1-HA and mCherry</li></ul>
 		</div>
 		<div class="col-sm-6">
@@ Line 111: / Line 111: @@
 	<div class="row category-row">
 		<div class="col-sm-6">
-			<p>-	mCAT-1 tagged with a C-terminal HA and mCherry</p>
+		<ul><li>mCAT-1 tagged with a C-terminal HA and mCherry</li>
-			<p>-	the HA and the mCherry are linked by … resulting in a doubled tagged mCAT-1 fusion protein</p>
+		    <li>the HA and the mCherry are linked by … resulting in a doubled tagged mCAT-1 fusion protein</li></ul>
 		</div>
 		<div class="col-sm-6">
@@ Line 128: / Line 128: @@
 	<div class="row category-row">
 		<div class="col-sm-6">
-			<p>-	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)</p>
+		<ul><li>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)</li>
-			<p>-	the HA and the mCherry are separated by a P2A sequence that results in 2 separate proteins, mCAT-1-HA and mCherry</p>
+			<li>the HA and the mCherry are separated by a P2A sequence that results in 2 separate proteins, mCAT-1-HA and mCherry</li></ul>
 		</div>
 		<div class="col-sm-6">
@@ Line 142: / Line 142: @@
 	<div class="row category-row">
 		<div class="col-sm-6">
-			<p>-	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)</p>
+		<ul><li>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)</li>
-			<p>-	the HA and the mCherry are linked by … resulting in a doubled tagged mCAT-1 fusion protein</p>
+		    <li>the HA and the mCherry are linked by … resulting in a doubled tagged mCAT-1 fusion protein</li></ul>
 		</div>
 		<div class="col-sm-6">
@@ Line 159: / Line 159: @@
 	<div class="row category-row">
 		<div class="col-sm-6">
-			<p>-	inducible expression vector encoding mCAT-1 under control of a galactose-inducible promoter (Gal4UAS)</p>
+		<ul><li>inducible expression vector encoding mCAT-1 under control of a galactose-inducible promoter (Gal4UAS)</li>
-			<p>-	in the dark the expression of mCAT-1 gene is off</p>
+		    <li>in the dark the expression of mCAT-1 gene is off</li>
-                        <p>-	to induce expression of the mCAT-1 gene the … plasmid is/are needed</p>
+                    <li>to induce expression of the mCAT-1 gene the … plasmid is/are needed</li></ul>
 		</div>
 		<div class="col-sm-6">
@@ Line 174: / Line 174: @@
 	<div class="row category-row">
 		<div class="col-sm-6">
-			<p>-	inducible expression vector encoding the mCAT-1 fusion protein (tagged with a C-terminal HA) under control of a galactose-inducible promoter (Gal4UAS)</p>
+		<ul><li>inducible expression vector encoding the mCAT-1 fusion protein (tagged with a C-terminal HA) under control of a galactose-inducible promoter (Gal4UAS)</li>
-			<p>-	in the dark the expression of mCAT-1 gene is off</p>
+			<li>in the dark the expression of mCAT-1 gene is off</li>
-                         <p>-	to induce expression of the mCAT-1 gene the … plasmid is/are needed</p>
+                         <li>to induce expression of the mCAT-1 gene the … plasmid is/are needed</li></ul>
 		</div>
 		<div class="col-sm-6">
@@ Line 202: / Line 202: @@
 	<div class="row category-row">
 		<div class="col-sm-6">
-			<p>-	bistronic vector encoding  COP1–VP16 and TetR-UVR8 under control a SV40 promoter
+		<ul><li>bistronic vector encoding  COP1–VP16 and TetR-UVR8 under control a SV40 promoter
 o	E3 ubiquitin-protein ligase COP1
 o	herpes simplex virus (HSV) transactivator protein VP16
-</p>
+</li></ul>
 		</div>
 		<div class="col-sm-6">
@@ Line 218: / Line 218: @@
 	<div class="row category-row">
 		<div class="col-sm-6">
-			<p>-	inducible expression vector encoding the reporter gene mCherry under control of a galactose-inducible promoter (Gal4UAS)</p>
+		<ul><li>inducible expression vector encoding the reporter gene mCherry under control of a galactose-inducible promoter (Gal4UAS)</li>
-			<p>-	in the dark the expression of the reporter gene is off</p>
+		    <li>in the dark the expression of the reporter gene is off</li>
-                        <p>-	to induce expression of mCherry the … plasmid is/are needed</p>
+                    <li>to induce expression of mCherry the … plasmid is/are needed</li>
 		</div>
 		<div class="col-sm-6">

Team:Freiburg/Content/Results/BioBrick