http://2007.igem.org/wiki/index.php?title=Special:Contributions/Klapoetke&feed=atom&limit=50&target=Klapoetke&year=&month=2007.igem.org - User contributions [en]2024-03-28T09:55:10ZFrom 2007.igem.orgMediaWiki 1.16.5http://2007.igem.org/wiki/index.php/Wisconsin/ProjectWisconsin/Project2007-10-27T00:52:09Z<p>Klapoetke: </p>
<hr />
<div>===Project Overview===<br />
Reprogramming gene expression is one of the main goals of synthetic biology. In the past, most groups have focused on cis-regulatory elements such as promoter to control transcription. We decided to explore artificial transcription factor (ATF), a trans-regulatory element, for controlling genes. Our project consists of characterizing ATF and testing ATF in mammalian cells.<br />
<br />
==Artificial Transcription Factor==<br />
===Background===<br />
Artificial transcription factor (ATF) has two domains: DNA-binding domain and regulatory domain. The DNA-binding domain provides sequence specific targeting and the regulatory domain can up- or down-regulate gene expression. ATF design is modular, meaning one can mix and match different DNA-binding motifs with various transcription factors.<br />
<br />
We decided to use zinc fingers for the DNA-binding domain. A zinc finger is made of about 30 amino acids and is stabilized by a single zinc ion to form a certain protein structure (eg. beta-beta-alpha). It can interact with a specific 3 base-pair motif in DNA (Segal & Barbas, 2001). Regulation of gene activity is achieved by using zinc finger to recognize and bind to a specific DNA sequences, and then recruite other molecular machinery that will activate or repress the gene. Our ATF project has focused on characterizing DNA binding specificity and cooperativity effects.<br />
<br />
===Design===<br />
Our ATF design has 3 zinc fingers, each can recognize 3bp of DNA sequence. So it should bind to a unique 9bp sequence. Following the zinc fingers are NLS and VP64. NLS is for localizing ATF to the nucleus and VP64 is a strong transcription activator. In order to test DNA-binding specificity, we also performed site-directed mutagenesis to change the zinc finger 1's recognition sequence from GAA to GGG (as shown below). This way we can test how well similar zinc fingers can discriminate DNA sequences. <br />
<br />
[[image:zif.jpg]]<br />
<br />
===Cognate Site Identity (CSI) Microarray===<br />
We are using CSI microarrays to quantify how well zinc fingers bind to DNA sequences. The CSI array we designed contains every permutation of 9bp DNA sequence. This allows us to test the entire sequence space in an unbiased manner. Below is a preliminary CSI array scan of purified ATF protein on an 8mer CSI array. <br />
<br />
[[Image:csiarray.jpg]]<br />
<br />
==BCL-2 and Cell Fate Regulation==<br />
===Background===<br />
Doxrubicin is a widely used drug to treat cancer. However one of its main limitation is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to use ATF to up-regulate BCL-2 (a pro-survival protein) and prevent cells from dying under mild stress. <br />
<br />
===Design===<br />
We designed a zinc finger ATF that will bind to BCL-2 promoter region and up-regulate transcription. The mammalian expression vector we used is tetracycline controlled. Adding tetracycline will induce ATF expression.<br />
<br />
[[Image:tetsys.jpg]]<br />
<br />
===Experiment===<br />
We tested our construct for function in NIH 3T3 (mouse fibroblast) cells. The first part of our experiment is to check for ATF and BCL-2 expression. We did this by looking at western blots. We successfully transfected our ATF into fibroblast cells but we were unable to detect any ATF proteins. We tried using different concentration of tetracycline but that did not improve our results. Since ATF was not expressed, the BCL-2 levels remained constant under all conditions. This is expected since BCL-2 is constitutively expressed under normal conditions. Unfortunately we were not able to test how BCL-2 levels affect cell fate regulation.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/ProjectWisconsin/Project2007-10-26T22:48:28Z<p>Klapoetke: /* Cognate Site Identity (CSI) Microarray */</p>
<hr />
<div>===Project Overview===<br />
Reprogramming gene expression is one of the main goals of synthetic biology. In the past, most groups have focused on cis-regulatory elements such as promoter to control transcription. We decided to explore artificial transcription factor (ATF), a trans-regulatory element, for controlling genes. Our project consists of characterizing ATF and testing ATF in mammalian cells.<br />
<br />
==Artificial Transcription Factor==<br />
===Background===<br />
Artificial transcription factor (ATF) has two domains: DNA-binding domain and regulatory domain. The DNA-binding domain provides sequence specific targeting and the regulatory domain can up- or down-regulate gene expression. ATF design is modular, meaning one can mix and match different DNA-binding motifs with various transcription factors.<br />
<br />
We decided to use zinc fingers for DNA-binding. A zinc finger is made of about 30 amino acids and is stabilized by a single zinc ion to form a certain protein structure (eg. beta-beta-alpha). It can interact with a specific 3 base-pair motif in DNA (Segal & Barbas, 2001). Regulation of gene activity is achieved by using zinc finger to recognize a specific DNA sequences, bind to the DNA, and recruite other molecular machinery that will activate or repress the gene. Our ATF project has centered on characterizing DNA binding specificity and cooperativity effects.<br />
<br />
===Design===<br />
Our ATF design has 3 zinc fingers followed NLS and VP64. NLS is for localizing ATF to the nucleus and VP64 is a strong transcription activator. We also performed site-directed mutagenesis to change the zinc finger recognition sequence.<br />
<br />
[[image:zif.jpg]]<br />
<br />
===Cognate Site Identity (CSI) Microarray===<br />
We are using CSI microarrays to quantify how well zinc fingers bind to DNA sequences. The CSI array contains every permutation of 9bp DNA sequence. This allows us to test the entire sequence space in an unbiased manner.<br />
<br />
[[Image:csiarray.jpg]]<br />
<br />
Preliminary scan of ATF on 8mer CSI array.<br />
<br />
==BCL-2 and Cell Fate Regulation==<br />
===Background===<br />
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. It also offers a good system for conducting experiments in vivo through its involvement in cellular disease states, particularly in reference to cellular mitochondria. Our goal is to up-regulate BCL-2 (a pro-survival protein) and prevent cells from dying under mild stress.<br />
<br />
===Design===<br />
Using a zinc-finger Artificial Transcription Factor (ATF) backbone, we designed an in vivo gene regulation system of Bcl-2. We prepared a highly developed ATF that is programmed to bind the promoter region of Bcl-2 and is subject to control by tetracycline.<br />
<br />
[[Image:tetsys.jpg]]<br />
<br />
===Experiment===<br />
We tested our construct for function in mammalian cells. Experiments were conducted by introducing our ATF into mouse fibroblast cells through transfection, adding tetracycline in certain concentrations, and then measuring protein levels within the cells. Our project design predicts that cells that have the ATF and receive a dose of tetracycline should have increased Bcl-2 gene expression. We would then be able to detect this up-regulated gene activity by measuring the subsequent increase production of Bcl-2 protein by western blot.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/ProjectWisconsin/Project2007-10-26T22:48:19Z<p>Klapoetke: /* Cognate Site Identity (CSI) Microarray */</p>
<hr />
<div>===Project Overview===<br />
Reprogramming gene expression is one of the main goals of synthetic biology. In the past, most groups have focused on cis-regulatory elements such as promoter to control transcription. We decided to explore artificial transcription factor (ATF), a trans-regulatory element, for controlling genes. Our project consists of characterizing ATF and testing ATF in mammalian cells.<br />
<br />
==Artificial Transcription Factor==<br />
===Background===<br />
Artificial transcription factor (ATF) has two domains: DNA-binding domain and regulatory domain. The DNA-binding domain provides sequence specific targeting and the regulatory domain can up- or down-regulate gene expression. ATF design is modular, meaning one can mix and match different DNA-binding motifs with various transcription factors.<br />
<br />
We decided to use zinc fingers for DNA-binding. A zinc finger is made of about 30 amino acids and is stabilized by a single zinc ion to form a certain protein structure (eg. beta-beta-alpha). It can interact with a specific 3 base-pair motif in DNA (Segal & Barbas, 2001). Regulation of gene activity is achieved by using zinc finger to recognize a specific DNA sequences, bind to the DNA, and recruite other molecular machinery that will activate or repress the gene. Our ATF project has centered on characterizing DNA binding specificity and cooperativity effects.<br />
<br />
===Design===<br />
Our ATF design has 3 zinc fingers followed NLS and VP64. NLS is for localizing ATF to the nucleus and VP64 is a strong transcription activator. We also performed site-directed mutagenesis to change the zinc finger recognition sequence.<br />
<br />
[[image:zif.jpg]]<br />
<br />
===Cognate Site Identity (CSI) Microarray===<br />
We are using CSI microarrays to quantify how well zinc fingers bind to DNA sequences. The CSI array contains every permutation of 9bp DNA sequence. This allows us to test the entire sequence space in an unbiased manner.<br />
<br />
[[Image:csiarray.jpg]]<br />
Preliminary scan of ATF on 8mer CSI array.<br />
<br />
==BCL-2 and Cell Fate Regulation==<br />
===Background===<br />
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. It also offers a good system for conducting experiments in vivo through its involvement in cellular disease states, particularly in reference to cellular mitochondria. Our goal is to up-regulate BCL-2 (a pro-survival protein) and prevent cells from dying under mild stress.<br />
<br />
===Design===<br />
Using a zinc-finger Artificial Transcription Factor (ATF) backbone, we designed an in vivo gene regulation system of Bcl-2. We prepared a highly developed ATF that is programmed to bind the promoter region of Bcl-2 and is subject to control by tetracycline.<br />
<br />
[[Image:tetsys.jpg]]<br />
<br />
===Experiment===<br />
We tested our construct for function in mammalian cells. Experiments were conducted by introducing our ATF into mouse fibroblast cells through transfection, adding tetracycline in certain concentrations, and then measuring protein levels within the cells. Our project design predicts that cells that have the ATF and receive a dose of tetracycline should have increased Bcl-2 gene expression. We would then be able to detect this up-regulated gene activity by measuring the subsequent increase production of Bcl-2 protein by western blot.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/File:Csiarray.jpgFile:Csiarray.jpg2007-10-26T22:48:14Z<p>Klapoetke: </p>
<hr />
<div></div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/ProjectWisconsin/Project2007-10-26T21:59:25Z<p>Klapoetke: /* Design */</p>
<hr />
<div>===Project Overview===<br />
Reprogramming gene expression is one of the main goals of synthetic biology. In the past, most groups have focused on cis-regulatory elements such as promoter to control transcription. We decided to explore artificial transcription factor (ATF), a trans-regulatory element, for controlling genes. Our project consists of characterizing ATF and testing ATF in mammalian cells.<br />
<br />
==Artificial Transcription Factor==<br />
===Background===<br />
Artificial transcription factor (ATF) has two domains: DNA-binding domain and regulatory domain. The DNA-binding domain provides sequence specific targeting and the regulatory domain can up- or down-regulate gene expression. ATF design is modular, meaning one can mix and match different DNA-binding motifs with various transcription factors.<br />
<br />
We decided to use zinc fingers for DNA-binding. A zinc finger is made of about 30 amino acids and is stabilized by a single zinc ion to form a certain protein structure (eg. beta-beta-alpha). It can interact with a specific 3 base-pair motif in DNA (Segal & Barbas, 2001). Regulation of gene activity is achieved by using zinc finger to recognize a specific DNA sequences, bind to the DNA, and recruite other molecular machinery that will activate or repress the gene. Our ATF project has centered on characterizing DNA binding specificity and cooperativity effects.<br />
<br />
===Design===<br />
Our ATF design has 3 zinc fingers followed NLS and VP64. NLS is for localizing ATF to the nucleus and VP64 is a strong transcription activator. We also performed site-directed mutagenesis to change the zinc finger recognition sequence.<br />
<br />
[[image:zif.jpg]]<br />
<br />
===Cognate Site Identity (CSI) Microarray===<br />
We are using CSI microarrays to quantify how well zinc fingers bind to DNA sequences. The CSI array contains every permutation of 9bp DNA sequence. This allows us to test the entire sequence space in an unbiased manner.<br />
<br />
==BCL-2 and Cell Fate Regulation==<br />
===Background===<br />
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to up-regulate BCL-2 (a pro-survival protein) and prevent cells from dying under mild stress.<br />
<br />
===Design===<br />
Using a zinc-finger Artificial Transcription Factor (ATF) backbone, we designed an in vivo gene regulation system of Bcl-2. This gene was chosen due to its transcription of an empirically important protein; it also offers a good system for conducting experiments in vivo through its involvement in cellular disease states, particularly in reference to cellular mitochondria. We prepared a highly developed ATF that is programmed to bind the promoter region of Bcl-2 and is subject to control by tetracycline.<br />
<br />
[[Image:tetsys.jpg]]<br />
<br />
===Experiment===<br />
We tested our construct for function in mammalian cells. The experiments were conducted by introducing our ATF into mouse fibroblast cells, adding tetracycline in certain conditions, and then measuring protein levels made by the cells. Our project design predicts that cells that have the ATF and receive a dose of tetracycline should have increased Bcl-2 gene expression. We would then be able to detect this up-regulated gene activity by measuring the subsequent increase production of Bcl-2 protein by western blot.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/ProjectWisconsin/Project2007-10-26T21:49:23Z<p>Klapoetke: /* Background */</p>
<hr />
<div>===Project Overview===<br />
Reprogramming gene expression is one of the main goals of synthetic biology. In the past, most groups have focused on cis-regulatory elements such as promoter to control transcription. We decided to explore artificial transcription factor (ATF), a trans-regulatory element, for controlling genes. Our project consists of characterizing ATF and testing ATF in mammalian cells.<br />
<br />
==Artificial Transcription Factor==<br />
===Background===<br />
Artificial transcription factor (ATF) has two domains: DNA-binding domain and regulatory domain. The DNA-binding domain provides sequence specific targeting and the regulatory domain can up- or down-regulate gene expression. ATF design is modular, meaning one can mix and match different DNA-binding motifs with various transcription factors.<br />
<br />
We decided to use zinc fingers for DNA-binding. A zinc finger is made of about 30 amino acids and is stabilized by a single zinc ion to form a certain protein structure (eg. beta-beta-alpha). It can interact with a specific 3 base-pair motif in DNA (Segal & Barbas, 2001). Regulation of gene activity is achieved by using zinc finger to recognize a specific DNA sequences, bind to the DNA, and recruite other molecular machinery that will activate or repress the gene. Our ATF project has centered on characterizing DNA binding specificity and cooperativity effects.<br />
<br />
===Design===<br />
We designed an ATF that can recognize 9 base pairs and characterized it with CSI array.<br />
[[image:zif.jpg]]<br />
<br />
===Cognate Site Identity (CSI) Microarray===<br />
We are using CSI microarrays to quantify how well zinc fingers bind to DNA sequences. The CSI array contains every permutation of 9bp DNA sequence. This allows us to test the entire sequence space in an unbiased manner.<br />
<br />
==BCL-2 and Cell Fate Regulation==<br />
===Background===<br />
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to up-regulate BCL-2 (a pro-survival protein) and prevent cells from dying under mild stress.<br />
<br />
===Design===<br />
Using zinc-finger concepts, we designed an in vivo gene regulation system. We chose to target the Bcl-2 gene, because it makes an empirically important protein, and offers a good system for conducting experiments in vivo by its involvement in cellular disease states. We prepared a highly developed ATF, one that is programmed to bind the promoter region of Bcl-2 and is subject to control by tetracycline itself.<br />
<br />
[[Image:tetsys.jpg]]<br />
<br />
===Experiment===<br />
We tested our construct for function in mammalian cells. The experiments were conducted by introducing our ATF into mouse fibroblast cells, adding tetracycline in certain conditions, and then measuring protein levels made by the cells. Our project design predicts that cells that have the ATF and receive a dose of tetracycline should have increased Bcl-2 gene expression. We would then be able to detect this up-regulated gene activity by measuring the subsequent increase production of Bcl-2 protein by western blot.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/ProjectWisconsin/Project2007-10-26T21:34:38Z<p>Klapoetke: /* Design */</p>
<hr />
<div>===Project Overview===<br />
Reprogramming gene expression is one of the main goals of synthetic biology. In the past, most groups have focused on cis-regulatory elements such as promoter to control transcription. We decided to explore artificial transcription factor (ATF), a trans-regulatory element, for controlling genes. Our project consists of characterizing ATF and testing ATF in mammalian cells.<br />
<br />
==Artificial Transcription Factor==<br />
===Background===<br />
Artificial transcription factor (ATF) has two domains: DNA-binding domain and regulatory domain. The DNA-binding domain provides sequence specific targeting and the regulatory domain can up- or down-regulate gene expression. ATF design is modular, meaning one can mix and match different DNA-binding motifs with various transcription factors.<br />
<br />
We decided to use zinc fingers for DNA-binding. In general, a zinc finger is 30 amino acids long, is stabilized by a single zinc ion to form a certain protein structure (beta-beta-alpha), and can interact with a specific 3 base-pair motif in DNA (Segal & Barbas, 2001). Regulation of gene activity is achieved by a ZFP recognizing a specific DNA sequences, binding to it, and recruiting other molecular machinery that will activate or repress the gene being targeted. It is this ability to target specific sites on DNA and then induce cellular activity in a controlled and predictable manner that is causing such anticipation. Researchers are learning to manipulate zinc-fingers artificially, so that they can be used to control gene expression in a meaningful way (Ansari, 2003, Segal et al., 1999). Our iGEM project has centered on further developing these ideas with ZFP's, and experimenting with real world applications, in particular to use ZFP’s as an artificial transcription factor (ATF). This would, we hope, create a powerful, new class of Bio-brick.<br />
<br />
===Design===<br />
We designed an ATF that can recognize 9 base pairs and characterized it with CSI array.<br />
<br />
===Cognate Site Identity (CSI) Microarray===<br />
We are using CSI microarrays to quantify how well zinc fingers bind to DNA sequences. The CSI array contains every permutation of 9bp DNA sequence. This allows us to test the entire sequence space in an unbiased manner.<br />
<br />
==BCL-2 and Cell Fate Regulation==<br />
===Background===<br />
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to up-regulate BCL-2 (a pro-survival protein) and prevent cells from dying under mild stress.<br />
<br />
===Design===<br />
Using zinc-finger concepts, we designed an in vivo gene regulation system. We chose to target the Bcl-2 gene, because it makes an empirically important protein, and offers a good system for conducting experiments in vivo by its involvement in cellular disease states. We prepared a highly developed ATF, one that is programmed to bind the promoter region of Bcl-2 and is subject to control by tetracycline itself.<br />
<br />
[[Image:tetsys.jpg]]<br />
<br />
===Experiment===<br />
We tested our construct for function in mammalian cells. The experiments were conducted by introducing our ATF into mouse fibroblast cells, adding tetracycline in certain conditions, and then measuring protein levels made by the cells. Our project design predicts that cells that have the ATF and receive a dose of tetracycline should have increased Bcl-2 gene expression. We would then be able to detect this up-regulated gene activity by measuring the subsequent increase production of Bcl-2 protein by western blot.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/File:Tetsys.jpgFile:Tetsys.jpg2007-10-26T21:28:26Z<p>Klapoetke: </p>
<hr />
<div></div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-10-26T20:59:36Z<p>Klapoetke: </p>
<hr />
<div>{| border="0" cellspacing="6px" cellpadding="10" width="100%" <br />
|-<br />
|align="center"| [[Wisconsin|Home]]<br />
|align="center"|[[Wisconsin/Project|Project]]<br />
|align="center"|[[Wisconsin/Protocol|Protocols]]<br />
|align="center"|[[Wisconsin/Acknowledgements|Sponsors]]<br />
|align="center"|[[Wisconsin/About|About Us]]<br />
|}<br />
<br />
<center>[[Image:IGEM_soft_logo.jpg]]</center></div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/AboutWisconsin/About2007-10-26T20:58:23Z<p>Klapoetke: /* Widgeteers */</p>
<hr />
<div>==Widgeteers==<br />
iGEM Team<br />
*Nathan Klapoetke<br />
*Sean McMaster<br />
*David Peterson<br />
<br />
Advisors<br />
*Clay Carlson<br />
*Leslie Donato<br />
*Mary Ozers<br />
<br />
Faculty<br />
*Franco Cerrina<br />
*Michael Sussman<br />
*Aseem Ansari<br />
*Doug Weibel</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/AcknowledgementsWisconsin/Acknowledgements2007-10-26T20:55:10Z<p>Klapoetke: </p>
<hr />
<div>We would like to thank the College of Engineering, Nanoscale Science and Engineering Center, and Chancellor John Wiley for their financial support.<br />
<br />
[[Image:uwmadison-coe.jpg]]<br />
<br />
[[Image:uw_nsec_header.gif]]</div>Klapoetkehttp://2007.igem.org/wiki/index.php/File:Uw_nsec_header.gifFile:Uw nsec header.gif2007-10-26T20:54:41Z<p>Klapoetke: </p>
<hr />
<div></div>Klapoetkehttp://2007.igem.org/wiki/index.php/File:Uwmadison-coe.jpgFile:Uwmadison-coe.jpg2007-10-26T20:53:55Z<p>Klapoetke: </p>
<hr />
<div></div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/ProjectWisconsin/Project2007-10-26T20:44:21Z<p>Klapoetke: /* Cognate Site Identity (CSI) Microarray */</p>
<hr />
<div>===Project Overview===<br />
Reprogramming gene expression is one of the main goals of synthetic biology. In the past, most groups have focused on cis-regulatory elements such as promoter to control transcription. We decided to explore artificial transcription factor (ATF), a trans-regulatory element, for controlling genes. Our project consists of characterizing ATF and testing ATF in mammalian cells.<br />
<br />
==Artificial Transcription Factor==<br />
===Background===<br />
Artificial transcription factor (ATF) has two domains: DNA-binding domain and regulatory domain. The DNA-binding domain provides sequence specific targeting and the regulatory domain can up- or down-regulate gene expression. ATF design is modular, meaning one can mix and match different DNA-binding motifs with various transcription factors.<br />
<br />
We decided to use zinc fingers for DNA-binding. In general, a zinc finger is 30 amino acids long, is stabilized by a single zinc ion to form a certain protein structure (beta-beta-alpha), and can interact with a specific 3 base-pair motif in DNA (Segal & Barbas, 2001). Regulation of gene activity is achieved by a ZFP recognizing a specific DNA sequences, binding to it, and recruiting other molecular machinery that will activate or repress the gene being targeted. It is this ability to target specific sites on DNA and then induce cellular activity in a controlled and predictable manner that is causing such anticipation. Researchers are learning to manipulate zinc-fingers artificially, so that they can be used to control gene expression in a meaningful way (Ansari, 2003, Segal et al., 1999). Our iGEM project has centered on further developing these ideas with ZFP's, and experimenting with real world applications, in particular to use ZFP’s as an artificial transcription factor (ATF). This would, we hope, create a powerful, new class of Bio-brick.<br />
<br />
===Design===<br />
We designed an ATF that can recognize 9 base pairs and characterized it with CSI array.<br />
<br />
===Cognate Site Identity (CSI) Microarray===<br />
We are using CSI microarrays to quantify how well zinc fingers bind to DNA sequences. The CSI array contains every permutation of 9bp DNA sequence. This allows us to test the entire sequence space in an unbiased manner.<br />
<br />
==BCL-2 and Cell Fate Regulation==<br />
===Background===<br />
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to up-regulate BCL-2 (a pro-survival protein) and prevent cells from dying under mild stress.<br />
<br />
===Design===<br />
Using zinc-finger concepts, we designed an in vivo gene regulation system. We chose to target the Bcl-2 gene, because it makes an empirically important protein, and offers a good system for conducting experiments in vivo by its involvement in cellular disease states. We prepared a highly developed ATF, one that is programmed to bind the promoter region of Bcl-2 and is subject to control by tetracycline itself. <br />
<br />
===Experiment===<br />
We tested our construct for function in mammalian cells. The experiments were conducted by introducing our ATF into mouse fibroblast cells, adding tetracycline in certain conditions, and then measuring protein levels made by the cells. Our project design predicts that cells that have the ATF and receive a dose of tetracycline should have increased Bcl-2 gene expression. We would then be able to detect this up-regulated gene activity by measuring the subsequent increase production of Bcl-2 protein by western blot.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/ProjectWisconsin/Project2007-10-26T20:44:01Z<p>Klapoetke: /* Cognate Site Identity (CSI) Array */</p>
<hr />
<div>===Project Overview===<br />
Reprogramming gene expression is one of the main goals of synthetic biology. In the past, most groups have focused on cis-regulatory elements such as promoter to control transcription. We decided to explore artificial transcription factor (ATF), a trans-regulatory element, for controlling genes. Our project consists of characterizing ATF and testing ATF in mammalian cells.<br />
<br />
==Artificial Transcription Factor==<br />
===Background===<br />
Artificial transcription factor (ATF) has two domains: DNA-binding domain and regulatory domain. The DNA-binding domain provides sequence specific targeting and the regulatory domain can up- or down-regulate gene expression. ATF design is modular, meaning one can mix and match different DNA-binding motifs with various transcription factors.<br />
<br />
We decided to use zinc fingers for DNA-binding. In general, a zinc finger is 30 amino acids long, is stabilized by a single zinc ion to form a certain protein structure (beta-beta-alpha), and can interact with a specific 3 base-pair motif in DNA (Segal & Barbas, 2001). Regulation of gene activity is achieved by a ZFP recognizing a specific DNA sequences, binding to it, and recruiting other molecular machinery that will activate or repress the gene being targeted. It is this ability to target specific sites on DNA and then induce cellular activity in a controlled and predictable manner that is causing such anticipation. Researchers are learning to manipulate zinc-fingers artificially, so that they can be used to control gene expression in a meaningful way (Ansari, 2003, Segal et al., 1999). Our iGEM project has centered on further developing these ideas with ZFP's, and experimenting with real world applications, in particular to use ZFP’s as an artificial transcription factor (ATF). This would, we hope, create a powerful, new class of Bio-brick.<br />
<br />
===Design===<br />
We designed an ATF that can recognize 9 base pairs and characterized it with CSI array.<br />
<br />
===Cognate Site Identity (CSI) Microarray===<br />
We used CSI microarrays to quantify how well zinc fingers bind to DNA sequences. The CSI array contains every permutation of 9bp DNA sequence. This allows us to test the entire sequence space in an unbiased manner.<br />
<br />
==BCL-2 and Cell Fate Regulation==<br />
===Background===<br />
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to up-regulate BCL-2 (a pro-survival protein) and prevent cells from dying under mild stress.<br />
<br />
===Design===<br />
Using zinc-finger concepts, we designed an in vivo gene regulation system. We chose to target the Bcl-2 gene, because it makes an empirically important protein, and offers a good system for conducting experiments in vivo by its involvement in cellular disease states. We prepared a highly developed ATF, one that is programmed to bind the promoter region of Bcl-2 and is subject to control by tetracycline itself. <br />
<br />
===Experiment===<br />
We tested our construct for function in mammalian cells. The experiments were conducted by introducing our ATF into mouse fibroblast cells, adding tetracycline in certain conditions, and then measuring protein levels made by the cells. Our project design predicts that cells that have the ATF and receive a dose of tetracycline should have increased Bcl-2 gene expression. We would then be able to detect this up-regulated gene activity by measuring the subsequent increase production of Bcl-2 protein by western blot.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/ProjectWisconsin/Project2007-10-26T20:35:10Z<p>Klapoetke: /* Background */</p>
<hr />
<div>===Project Overview===<br />
Reprogramming gene expression is one of the main goals of synthetic biology. In the past, most groups have focused on cis-regulatory elements such as promoter to control transcription. We decided to explore artificial transcription factor (ATF), a trans-regulatory element, for controlling genes. Our project consists of characterizing ATF and testing ATF in mammalian cells.<br />
<br />
==Artificial Transcription Factor==<br />
===Background===<br />
Artificial transcription factor (ATF) has two domains: DNA-binding domain and regulatory domain. The DNA-binding domain provides sequence specific targeting and the regulatory domain can up- or down-regulate gene expression. ATF design is modular, meaning one can mix and match different DNA-binding motifs with various transcription factors.<br />
<br />
We decided to use zinc fingers for DNA-binding. In general, a zinc finger is 30 amino acids long, is stabilized by a single zinc ion to form a certain protein structure (beta-beta-alpha), and can interact with a specific 3 base-pair motif in DNA (Segal & Barbas, 2001). Regulation of gene activity is achieved by a ZFP recognizing a specific DNA sequences, binding to it, and recruiting other molecular machinery that will activate or repress the gene being targeted. It is this ability to target specific sites on DNA and then induce cellular activity in a controlled and predictable manner that is causing such anticipation. Researchers are learning to manipulate zinc-fingers artificially, so that they can be used to control gene expression in a meaningful way (Ansari, 2003, Segal et al., 1999). Our iGEM project has centered on further developing these ideas with ZFP's, and experimenting with real world applications, in particular to use ZFP’s as an artificial transcription factor (ATF). This would, we hope, create a powerful, new class of Bio-brick.<br />
<br />
===Design===<br />
We designed an ATF that can recognize 9 base pairs and characterized it with CSI array.<br />
<br />
===Cognate Site Identity (CSI) Array===<br />
<br />
<br />
==BCL-2 and Cell Fate Regulation==<br />
===Background===<br />
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to up-regulate BCL-2 (a pro-survival protein) and prevent cells from dying under mild stress.<br />
<br />
===Design===<br />
Using zinc-finger concepts, we designed an in vivo gene regulation system. We chose to target the Bcl-2 gene, because it makes an empirically important protein, and offers a good system for conducting experiments in vivo by its involvement in cellular disease states. We prepared a highly developed ATF, one that is programmed to bind the promoter region of Bcl-2 and is subject to control by tetracycline itself. <br />
<br />
===Experiment===<br />
We tested our construct for function in mammalian cells. The experiments were conducted by introducing our ATF into mouse fibroblast cells, adding tetracycline in certain conditions, and then measuring protein levels made by the cells. Our project design predicts that cells that have the ATF and receive a dose of tetracycline should have increased Bcl-2 gene expression. We would then be able to detect this up-regulated gene activity by measuring the subsequent increase production of Bcl-2 protein by western blot.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/ProjectWisconsin/Project2007-10-26T20:29:52Z<p>Klapoetke: </p>
<hr />
<div>===Project Overview===<br />
Reprogramming gene expression is one of the main goals of synthetic biology. In the past, most groups have focused on cis-regulatory elements such as promoter to control transcription. We decided to explore artificial transcription factor (ATF), a trans-regulatory element, for controlling genes. Our project consists of characterizing ATF and testing ATF in mammalian cells.<br />
<br />
==Artificial Transcription Factor==<br />
===Background===<br />
Artificial transcription factor (ATF) has two domains: DNA-binding domain and regulatory domain. The DNA-binding domain provides sequence specific targeting and the regulatory domain can up- or down-regulate gene expression. ATF design is modular, meaning one can mix and match different DNA-binding motifs with various transcription factors.<br />
<br />
We decided to use zinc fingers for DNA-binding. In general, a zinc finger is 30 amino acids long, is stabilized by a single zinc ion to form a certain protein structure (beta-beta-alpha), and can interact with a specific 3 base-pair motif in DNA (Segal & Barbas, 2001). Regulation of gene activity is achieved by a ZFP recognizing a specific DNA sequences, binding to it, and recruiting other molecular machinery that will activate or repress the gene being targeted. It is this ability to target specific sites on DNA and then induce cellular activity in a controlled and predictable manner that is causing such anticipation. Researchers are learning to manipulate zinc-fingers artificially, so that they can be used to control gene expression in a meaningful way (Ansari, 2003, Segal et al., 1999). Our iGEM project has centered on further developing these ideas with ZFP's, and experimenting with real world applications, in particular to use ZFP’s as an artificial transcription factor (ATF). This would, we hope, create a powerful, new class of Bio-brick.<br />
<br />
===Design===<br />
We designed an ATF that can recognize 9 base pairs and characterized it with CSI array.<br />
<br />
===Cognate Site Identity (CSI) Array===<br />
<br />
<br />
==BCL-2 and Cell Fate Regulation==<br />
===Background===<br />
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to up-regulate bcl-2 (a pro-survival protein) and prevent cells from dying under mild stress. We have designed an inducible artificial transcription factor (ATF) that binds to bcl-2 promoter. Currently we are studying cell fate regulation using ATF in mouse fibroblast cells.<br />
<br />
===Design===<br />
Using zinc-finger concepts, we designed an in vivo gene regulation system. We chose to target the Bcl-2 gene, because it makes an empirically important protein, and offers a good system for conducting experiments in vivo by its involvement in cellular disease states. We prepared a highly developed ATF, one that is programmed to bind the promoter region of Bcl-2 and is subject to control by tetracycline itself. <br />
<br />
===Experiment===<br />
We tested our construct for function in mammalian cells. The experiments were conducted by introducing our ATF into mouse fibroblast cells, adding tetracycline in certain conditions, and then measuring protein levels made by the cells. Our project design predicts that cells that have the ATF and receive a dose of tetracycline should have increased Bcl-2 gene expression. We would then be able to detect this up-regulated gene activity by measuring the subsequent increase production of Bcl-2 protein by western blot.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-10-26T20:29:10Z<p>Klapoetke: </p>
<hr />
<div>{| border="0" cellspacing="6px" cellpadding="10" width="100%" <br />
|-<br />
|align="center"| [[Wisconsin|Home]]<br />
|align="center"|[[Wisconsin/Project|Project]]<br />
|align="center"|[[Wisconsin/Protocol|Protocols]]<br />
|align="center"|[[Wisconsin/Acknowledgements|Acknowledgements]]<br />
|align="center"|[[Wisconsin/About|About Us]]<br />
|}<br />
<br />
<center>[[Image:IGEM_soft_logo.jpg]]</center></div>Klapoetkehttp://2007.igem.org/wiki/index.php/File:IGEM_soft_logo.jpgFile:IGEM soft logo.jpg2007-10-26T16:55:24Z<p>Klapoetke: </p>
<hr />
<div>[http://en.wikipedia.org/wiki/Image:Zinc_finger_DNA_complex.png Zinc finger] is rendered by Thomas Splettstoesser.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-10-26T16:40:45Z<p>Klapoetke: </p>
<hr />
<div>{| border="0" cellspacing="6px" cellpadding="10" width="100%" <br />
|-<br />
|align="center"| [[Wisconsin|Home]]<br />
|align="center"|[[Wisconsin/Project/ATF|ATF Project]]<br />
|align="center"|[[Wisconsin/Project/BCL2|BCL-2 Project]]<br />
|align="center"|[[Wisconsin/Protocol|Protocols]]<br />
|align="center"|[[Wisconsin/Acknowledgements|Acknowledgements]]<br />
|align="center"|[[Wisconsin/About|About Us]]<br />
|}<br />
<br />
<center>[[Image:IGEM_soft_logo.jpg]]</center></div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Project/BCL2Wisconsin/Project/BCL22007-10-26T16:12:26Z<p>Klapoetke: </p>
<hr />
<div>==BCL-2 and Cell Fate==<br />
===Background===<br />
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to up-regulate bcl-2 (a pro-survival protein) and prevent cells from dying under mild stress. We have designed an inducible artificial transcription factor (ATF) that binds to bcl-2 promoter. Currently we are studying cell fate regulation using ATF in mouse fibroblast cells.<br />
<br />
===Design===<br />
Using zinc-finger concepts, we designed an in vivo gene regulation system. We chose to target the Bcl-2 gene, because it makes an empirically important protein, and offers a good system for conducting experiments in vivo by its involvement in cellular disease states. We prepared a highly developed ATF, one that is programmed to bind the promoter region of Bcl-2 and is subject to control by tetracycline itself. <br />
<br />
===Experiment===<br />
We tested our construct for function in mammalian cells. The experiments were conducted by introducing our ATF into mouse fibroblast cells, adding tetracycline in certain conditions, and then measuring protein levels made by the cells. Our project design predicts that cells that have the ATF and receive a dose of tetracycline should have increased Bcl-2 gene expression. We would then be able to detect this up-regulated gene activity by measuring the subsequent increase production of Bcl-2 protein by western blot.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Project/ATFWisconsin/Project/ATF2007-10-26T16:09:35Z<p>Klapoetke: </p>
<hr />
<div>==Artificial Transcription Factor==<br />
'''What is Artificial Transcription Factor (ATF)?'''<br />
<br />
<br />
'''ATF Design'''<br />
<br />
<br />
'''CSI Array'''</div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-10-26T16:02:56Z<p>Klapoetke: </p>
<hr />
<div>{| border="0" cellspacing="6px" cellpadding="10" width="100%"<br />
|-<br />
|[[Wisconsin|Home]]<br />
|[[Wisconsin/Project/ATF|ATF Project]]<br />
|[[Wisconsin/Project/BCL2|BCL-2 Project]]<br />
|[[Wisconsin/Protocol|Protocols]]<br />
|[[Wisconsin/Acknowledgements|Acknowledgements]]<br />
|[[Wisconsin/About|About Us]]<br />
|}<br />
<br />
<center>[[Image:IGEM_soft_logo.jpg]]</center></div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/AboutWisconsin/About2007-10-26T16:01:13Z<p>Klapoetke: </p>
<hr />
<div>==Widgeteers==<br />
Junior Widgeteer<br />
*Nathan Klapoetke<br />
*Sean McMaster<br />
*David Peterson<br />
<br />
Chief Widgeteer<br />
*Aseem Ansari<br />
*Clay Carlson<br />
*Franco Cerrina<br />
*Mary Ozers<br />
*Doug Weibel</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/ProtocolWisconsin/Protocol2007-10-26T16:00:13Z<p>Klapoetke: </p>
<hr />
<div>==Protocol==<br />
<br />
===Cloning===<br />
*[[Wisconsin/Protocol:Digestion|Digestion]]<br />
*[[Wisconsin/Protocol:Ligation|Ligation]]<br />
*[[Wisconsin/Protocol:Miniprep|Miniprep]]<br />
*[[Wisconsin/Protocol:Primer|Primer Design]]<br />
*[[Wisconsin/Protocol:Transformation|Transformation]]<br />
*[[Wisconsin/Protocol:Cell Culture|Cell Culture]]<br />
<br />
===Instrument===<br />
*[[Wisconsin/Protocol:Agarose Gel|Gel Electrophoresis]]<br />
*[[Wisconsin/Protocol:NanoDrop|NanoDrop]]<br />
*[[Wisconsin/Protocol:PCR|PCR and PCR Purification]]</div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-10-26T15:59:02Z<p>Klapoetke: </p>
<hr />
<div>{| border="0" cellspacing="8px" cellpadding="15" width="100%"<br />
|-<br />
|[[Wisconsin|Home]]<br />
|[[Wisconsin/Project/ATF|ATF Project]]<br />
|[[Wisconsin/Project/BCL2|BCL-2 Project]]<br />
|[[Wisconsin/Protocol|Protocols]]<br />
|[[Wisconsin/Acknowledgements|Acknowledgements]]<br />
|[[Wisconsin/About|About Us]]<br />
|}<br />
<br />
<center>[[Image:IGEM_soft_logo.jpg]]</center><br />
<br />
==Widgeteers==<br />
Junior Widgeteer<br />
*Nathan Klapoetke<br />
*Sean McMaster<br />
*David Peterson<br />
<br />
<br />
Chief Widgeteer<br />
*Aseem Ansari<br />
*Clay Carlson<br />
*Franco Cerrina<br />
*Mary Ozers<br />
*Doug Weibel<br />
<br />
<br />
==[[Wisconsin/Project Description|Project ATF]]==<br />
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to up-regulate bcl-2 (a pro-survival protein) and prevent cells from dying under mild stress. We have designed an inducible artificial transcription factor (ATF) that binds to bcl-2 promoter. Currently we are studying cell fate regulation using ATF in mouse fibroblast cells.<br />
<br />
==Protocol==<br />
<br />
===Cloning===<br />
*[[Wisconsin/Protocol:Digestion|Digestion]]<br />
*[[Wisconsin/Protocol:Ligation|Ligation]]<br />
*[[Wisconsin/Protocol:Miniprep|Miniprep]]<br />
*[[Wisconsin/Protocol:Primer|Primer Design]]<br />
*[[Wisconsin/Protocol:Transformation|Transformation]]<br />
*[[Wisconsin/Protocol:Cell Culture|Cell Culture]]<br />
<br />
===Instrument===<br />
*[[Wisconsin/Protocol:Agarose Gel|Gel Electrophoresis]]<br />
*[[Wisconsin/Protocol:NanoDrop|NanoDrop]]<br />
*[[Wisconsin/Protocol:PCR|PCR and PCR Purification]]</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Project_DescriptionWisconsin/Project Description2007-08-15T14:46:55Z<p>Klapoetke: </p>
<hr />
<div>Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to up-regulate bcl-2 (a pro-survival protein) and prevent cells from dying under mild stress. We have designed an inducible artificial transcription factor (ATF) that binds to bcl-2 promoter. Currently we are studying cell fate regulation using ATF in mouse fibroblast cells.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-08-15T14:46:15Z<p>Klapoetke: /* Project ATF */</p>
<hr />
<div><center>[[Image:IGEM_soft_logo.jpg]]</center><br />
<br />
==Widgeteers==<br />
Junior Widgeteer<br />
*Nathan Klapoetke<br />
*Sean McMaster<br />
*David Peterson<br />
<br />
<br />
Chief Widgeteer<br />
*Aseem Ansari<br />
*Clay Carlson<br />
*Franco Cerrina<br />
*Mary Ozers<br />
*Doug Weibel<br />
<br />
<br />
==[[Wisconsin/Project Description|Project ATF]]==<br />
Doxrubicin is a widely used drug to treat cancer. However one main limitation of its use is cardiotoxicity. Accumulated dose of doxrubicin can cause oxidative stress to cardiomyocytes and trigger apoptosis. Our goal is to up-regulate bcl-2 (a pro-survival protein) and prevent cells from dying under mild stress. We have designed an inducible artificial transcription factor (ATF) that binds to bcl-2 promoter. Currently we are studying cell fate regulation using ATF in mouse fibroblast cells.<br />
<br />
==Protocol==<br />
<br />
===Cloning===<br />
*[[Wisconsin/Protocol:Digestion|Digestion]]<br />
*[[Wisconsin/Protocol:Ligation|Ligation]]<br />
*[[Wisconsin/Protocol:Miniprep|Miniprep]]<br />
*[[Wisconsin/Protocol:Primer|Primer Design]]<br />
*[[Wisconsin/Protocol:Transformation|Transformation]]<br />
*[[Wisconsin/Protocol:Cell Culture|Cell Culture]]<br />
<br />
===Instrument===<br />
*[[Wisconsin/Protocol:Agarose Gel|Gel Electrophoresis]]<br />
*[[Wisconsin/Protocol:NanoDrop|NanoDrop]]<br />
*[[Wisconsin/Protocol:PCR|PCR and PCR Purification]]</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:TransformationWisconsin/Protocol:Transformation2007-07-11T18:14:39Z<p>Klapoetke: /* Transforming Chemically Competent Cells */</p>
<hr />
<div>===Transforming Chemically Competent Cells===<br />
#Thaw competent cells on ice<br />
#Chill three 1.5mL tubes in ice bucket. One for transformation and the other two for control.<br />
#*Normal: pippet 100uL of cells and 9uL of ligation reaction into tube<br />
#*Positive Control: pippet 100uL of cells and 2uL of pUC18 or pUC19 into tube<br />
#*Negative Control: pippet 100uL of cells<br />
#Sit on ice for 30 minutes<br />
#Heat shock in 42<sup>o</sup>C water bath for 90 seconds<br />
#Incubate on ice for 2 minutes<br />
#Add 200uL of SOC to each culture tube<br />
#Incubate on shaker (nutator) at 37<sup>o</sup>C for 1 hour<br />
#Grow 4 plates: 1 normal diluted, 1 normal concentrated, and 2 controls concentrated.<br />
#*Diluted: Spread 50-100uL onto plate<br />
#*Concentrated: Centrifuge tube for 2-4 minutes at 5000rpm first, then spread everything except for 50-100uL onto plates.<br />
#Incubate plates for 16 hours at 37<sup>o</sup>C. Plates should face down to prevent condensation on surface.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:TransformationWisconsin/Protocol:Transformation2007-07-06T17:40:47Z<p>Klapoetke: /* Transforming Chemically Competent Cells */</p>
<hr />
<div>===Transforming Chemically Competent Cells===<br />
#Thaw cells on ice<br />
#Pippet 100uL of cells into three ice-cold 1.5mL tube. One for transformation and the other two for control.<br />
#*Normal: pippet 9uL of ligation reaction into tube<br />
#*Positive Control: pippet 2uL of pUC18 or pUC19 into tube<br />
#*Negative Control: nothing<br />
#Sit on ice for 30 minutes<br />
#Heat shock in 42<sup>o</sup>C water bath for 90 seconds<br />
#Incubate on ice for 2 minutes<br />
#Add 200uL of SOC to each culture tube<br />
#Incubate on shaker (nutator) at 37<sup>o</sup>C for 1 hour<br />
#Grow 4 plates: 1 normal diluted, 1 normal concentrated, and 2 controls concentrated.<br />
#*Diluted: Spread 50-100uL onto plate<br />
#*Concentrated: Centrifuge tube for 2-4 minutes at 5000rpm first, then spread everything except for 50-100uL onto plates.<br />
#Incubate plates for 16 hours at 37<sup>o</sup>C. Plates should face down to prevent condensation on surface.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:Agarose_GelWisconsin/Protocol:Agarose Gel2007-07-06T17:20:38Z<p>Klapoetke: /* 1.2% Agarose Gel preparation */</p>
<hr />
<div>===1.2% Agarose Gel preparation===<br />
*8 wells<br />
*600mg Agarose<br />
*50mL TBE<br />
*1uL 10mg/ml EtBr<br />
<br />
Mix agarose and TBE in flask, microwave for 20 second intervals and swirl it until solution is clear (usually 1-2min. total) to make sure agarose is dissolved in TBE. Add EtBr to flask (under hood) and pour into gel box with comb in place. Wait until gel solidifies.<br />
<br />
*2uL DNA<br />
*12uL ddH<sub>2</sub>O<br />
*4uL 5x Loading Dye<br />
*4uL DNA Ladder for reference<br />
<br />
Add water if DNA is less than 10uL and make sure #uL of 1x loading dye = #uL total. Mix content in tube before adding it to well. Also make sure gel box is submerged in TAE/TBE before adding DNA. Run to red half way.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:Agarose_GelWisconsin/Protocol:Agarose Gel2007-07-06T17:19:36Z<p>Klapoetke: /* 1.2% Agarose Gel preparation */</p>
<hr />
<div>===1.2% Agarose Gel preparation===<br />
*8 wells<br />
*600mg Agarose<br />
*50mL TBE<br />
*1uL 10mg/ml EtBr<br />
<br />
Mix agarose and TBE in flask, microwave for 20 second intervals and swirl it until solution is clear (usually 1-2min. total) to make sure agarose is dissolved in TBE. Add EtBr to flask (under hood) and pour into gel box with comb in place. Wait until gel solidifies.<br />
<br />
*2uL DNA<br />
*12uL ddH<sub>2</sub>O<br />
*4uL 5x Loading Dye<br />
*4uL DNA Ladder for reference<br />
<br />
Add water if DNA is less than 10uL and make sure #uL of 1x loading dye = #uL total. Mix content in tube before adding it to well. Also make sure gel box is submerged in TAE/TBE before adding DNA.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:LigationWisconsin/Protocol:Ligation2007-07-06T16:24:05Z<p>Klapoetke: /* Ligation */</p>
<hr />
<div>===Ligation===<br />
Use Nanodrop to find vector and insert concentration in [ng/uL]. Find the length of vector and insert and calculate volume of reagents needed with [http://mywebspace.wisc.edu/klapoetke/web/iGEM/WIDGET_Ligation_Reaction.xls this excel spreadsheet].<br />
<br />
Heat inactivate ligation afterwards: 15 minutes at 65C.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:TransformationWisconsin/Protocol:Transformation2007-07-06T16:23:19Z<p>Klapoetke: /* Transforming Chemically Competent Cells */</p>
<hr />
<div>===Transforming Chemically Competent Cells===<br />
#Thaw cells on ice<br />
#Pippet 100uL of cells into three ice-cold 1.5mL tube. One for transformation and the other two for control.<br />
#*Normal: pippet 9uL of ligation reaction into tube<br />
#*Positive Control: pippet 2uL of pUC18 or pUC19 into tube<br />
#*Negative Control: nothing<br />
#Sit on ice for 30 minutes<br />
#Heat shock in 42<sup>o</sup>C water bath for 90 seconds<br />
#Incubate on ice for 2 minutes<br />
#Add 200uL of SOC to each culture tube<br />
#Incubate on shaker (nutator) at 37<sup>o</sup>C for 1 hour<br />
#Grow 4 plates: 1 normal diluted, 1 normal concentrated, and 2 controls concentrated.<br />
#*Diluted: Spread 50-100uL onto plate<br />
#*Concentrated: Centrifuge tube for 2-4 minutes at 5000rpm first, then spread everything except for 200uL onto plates.<br />
#Incubate plates for 16 hours at 37<sup>o</sup>C. Plates should face down to prevent condensation on surface.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-07-06T00:52:55Z<p>Klapoetke: /* Instrument */</p>
<hr />
<div><center>[[Image:IGEM_soft_logo.jpg]]</center><br />
<br />
==Widgeteers==<br />
Junior Widgeteer<br />
*Nathan Klapoetke<br />
*Sean McMaster<br />
*David Peterson<br />
<br />
<br />
Chief Widgeteer<br />
*Aseem Ansari<br />
*Clay Carlson<br />
*Franco Cerrina<br />
*Mary Ozers<br />
*Doug Weibel<br />
<br />
<br />
==Project ATF==<br />
... more to come later<br />
<br />
<br />
==Protocol==<br />
<br />
===Cloning===<br />
*[[Wisconsin/Protocol:Digestion|Digestion]]<br />
*[[Wisconsin/Protocol:Ligation|Ligation]]<br />
*[[Wisconsin/Protocol:Miniprep|Miniprep]]<br />
*[[Wisconsin/Protocol:Primer|Primer Design]]<br />
*[[Wisconsin/Protocol:Transformation|Transformation]]<br />
<br />
===Instrument===<br />
*[[Wisconsin/Protocol:Agarose Gel|Gel Electrophoresis]]<br />
*[[Wisconsin/Protocol:NanoDrop|NanoDrop]]<br />
*[[Wisconsin/Protocol:PCR|PCR and PCR Purification]]</div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-07-05T23:29:08Z<p>Klapoetke: /* Instrument */</p>
<hr />
<div><center>[[Image:IGEM_soft_logo.jpg]]</center><br />
<br />
==Widgeteers==<br />
Junior Widgeteer<br />
*Nathan Klapoetke<br />
*Sean McMaster<br />
*David Peterson<br />
<br />
<br />
Chief Widgeteer<br />
*Aseem Ansari<br />
*Clay Carlson<br />
*Franco Cerrina<br />
*Mary Ozers<br />
*Doug Weibel<br />
<br />
<br />
==Project ATF==<br />
... more to come later<br />
<br />
<br />
==Protocol==<br />
<br />
===Cloning===<br />
*[[Wisconsin/Protocol:Digestion|Digestion]]<br />
*[[Wisconsin/Protocol:Ligation|Ligation]]<br />
*[[Wisconsin/Protocol:Miniprep|Miniprep]]<br />
*[[Wisconsin/Protocol:Primer|Primer Design]]<br />
*[[Wisconsin/Protocol:Transformation|Transformation]]<br />
<br />
===Instrument===<br />
*[[Wisconsin/Protocol:Agarose Gel|Gel Electrophoresis]]<br />
*[[Wisconsin/Protocol:PCR|PCR and PCR Purification]]</div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-07-05T23:28:47Z<p>Klapoetke: /* Instrument */</p>
<hr />
<div><center>[[Image:IGEM_soft_logo.jpg]]</center><br />
<br />
==Widgeteers==<br />
Junior Widgeteer<br />
*Nathan Klapoetke<br />
*Sean McMaster<br />
*David Peterson<br />
<br />
<br />
Chief Widgeteer<br />
*Aseem Ansari<br />
*Clay Carlson<br />
*Franco Cerrina<br />
*Mary Ozers<br />
*Doug Weibel<br />
<br />
<br />
==Project ATF==<br />
... more to come later<br />
<br />
<br />
==Protocol==<br />
<br />
===Cloning===<br />
*[[Wisconsin/Protocol:Digestion|Digestion]]<br />
*[[Wisconsin/Protocol:Ligation|Ligation]]<br />
*[[Wisconsin/Protocol:Miniprep|Miniprep]]<br />
*[[Wisconsin/Protocol:Primer|Primer Design]]<br />
*[[Wisconsin/Protocol:Transformation|Transformation]]<br />
<br />
===Instrument===<br />
[[Wisconsin/Protocol:Agarose Gel|Gel Electrophoresis]]<br />
<br />
[[Wisconsin/Protocol:PCR|PCR and PCR Purification]]</div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-07-05T23:28:37Z<p>Klapoetke: /* Protocol */</p>
<hr />
<div><center>[[Image:IGEM_soft_logo.jpg]]</center><br />
<br />
==Widgeteers==<br />
Junior Widgeteer<br />
*Nathan Klapoetke<br />
*Sean McMaster<br />
*David Peterson<br />
<br />
<br />
Chief Widgeteer<br />
*Aseem Ansari<br />
*Clay Carlson<br />
*Franco Cerrina<br />
*Mary Ozers<br />
*Doug Weibel<br />
<br />
<br />
==Project ATF==<br />
... more to come later<br />
<br />
<br />
==Protocol==<br />
<br />
===Cloning===<br />
*[[Wisconsin/Protocol:Digestion|Digestion]]<br />
*[[Wisconsin/Protocol:Ligation|Ligation]]<br />
*[[Wisconsin/Protocol:Miniprep|Miniprep]]<br />
*[[Wisconsin/Protocol:Primer|Primer Design]]<br />
*[[Wisconsin/Protocol:Transformation|Transformation]]<br />
<br />
===Instrument===<br />
[[Wisconsin/Protocol:Agarose Gel|Gel Electrophoresis]]<br />
[[Wisconsin/Protocol:PCR|PCR and PCR Purification]]</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:LigationWisconsin/Protocol:Ligation2007-07-05T23:27:57Z<p>Klapoetke: /* Ligation */</p>
<hr />
<div>===Ligation===<br />
Use Nanodrop to find vector and insert concentration in [ng/uL]. Find the length of vector and insert and calculate volume of reagents needed with [http://mywebspace.wisc.edu/klapoetke/web/iGEM/WIDGET_Ligation_Reaction.xls this excel spreadsheet].</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:TransformationWisconsin/Protocol:Transformation2007-07-05T23:05:41Z<p>Klapoetke: /* Transforming Chemically Competent Cells */</p>
<hr />
<div>===Transforming Chemically Competent Cells===<br />
#Thaw cells on ice<br />
#Pippet 100uL of cells into three 1.5mL tube. One for transformation and the other two for control.<br />
#*Normal: pippet 9uL of DNA into tube<br />
#*Positive Control: pippet 2uL of pUC18 or pUC19 into tube<br />
#*Negative Control: nothing<br />
#Sit on ice for 30 minutes<br />
#Heat shock in 42<sup>o</sup>C water bath for 30 seconds<br />
#Incubate on ice for 2 minutes<br />
#Transfer cells to new culture tubes (more O<sub>2</sub> for growth) and add 900uL of SOC to each culture tube<br />
#Incubate on shaker at 37<sup>o</sup>C for 1 hour<br />
#Grow 4 plates: 1 normal diluted, 1 normal concentrated, and 2 controls concentrated.<br />
#*Diluted: Spread 200uL onto plate<br />
#*Concentrated: Centrifuge tube for 5 minutes at 5000rpm first, then spread everything except for 200uL onto plates.<br />
#Incubate plates for 16 hours at 37<sup>o</sup>C. Plates should face down to prevent condensation on surface.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:TransformationWisconsin/Protocol:Transformation2007-07-05T23:05:02Z<p>Klapoetke: /* Transforming Chemically Competent Cells */</p>
<hr />
<div>===Transforming Chemically Competent Cells===<br />
#Thaw cells on ice<br />
#Pippet 100uL of cells into three 1.5mL tube. One for transformation and the other two for control.<br />
#*Normal: pippet 9uL of DNA into tube<br />
#*Positive Control: pippet 2uL of pUC18 or pUC19 into tube<br />
#*Negative Control: nothing<br />
#Sit on ice for 30 minutes<br />
#Heat shock in 42<sup>o</sup>C water bath for 30 seconds<br />
#Incubate on ice for 2 minutes<br />
#Transfer cells to new culture tubes (more O<sub>2</sub> for growth) and add 900uL of SOC to each culture tube<br />
#Incubate on shaker at 37<sup>o</sup>C for 1 hour<br />
#Grow 4 plates: 1 normal diluted, 1 normal concentrated, and 2 controls concentrated.<br />
#*Diluted: Spread 200uL onto plate<br />
#*Concentrated: Centrifuge all tubes for 5 minutes at 5000rpm first, then pippet everything except for 200uL onto plates.<br />
#Incubate plates for 16 hours at 37<sup>o</sup>C. Plates should face down to prevent condensation on surface.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:TransformationWisconsin/Protocol:Transformation2007-07-05T22:41:52Z<p>Klapoetke: /* Transforming Chemically Competent Cells */</p>
<hr />
<div>===Transforming Chemically Competent Cells===<br />
#Thaw cells on ice<br />
#Pippet 100uL of cells into three 1.5mL tube. One for transformation and the other two for control.<br />
#*Normal: pippet 9uL of DNA into tube<br />
#*Positive Control: pippet 2uL of pUC18 or pUC19 into tube<br />
#*Negative Control: nothing<br />
#Sit on ice for 30 minutes<br />
#Heat shock in 42<sup>o</sup>C water bath for 30 seconds<br />
#Incubate on ice for 2 minutes<br />
#Transfer cells to new culture tubes (more O<sub>2</sub> for growth) and add 900uL of SOC to each culture tube<br />
#Incubate on shaker at 37<sup>o</sup>C for 1 hour<br />
#Spread ??? uL onto plate... centrifuge first???<br />
#Incubate plates for 16 hours at 37<sup>o</sup>C. Put plates upside down to prevent condensation on surface.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:TransformationWisconsin/Protocol:Transformation2007-07-05T22:16:27Z<p>Klapoetke: </p>
<hr />
<div>===Transforming Chemically Competent Cells===<br />
#Thaw cells on ice<br />
#Pippet 100uL of cells into three 1.5mL tube. One for transformation and the other two for control.<br />
#*Normal: pippet 9uL of DNA into tube<br />
#*Positive Control: pippet 2uL of pUC18 or pUC19 into tube<br />
#*Negative Control: nothing<br />
#Sit on ice for 30 minutes<br />
#Heat shock in 42<sup>o</sup>C water bath for 30 seconds<br />
#Incubate on ice for 2 minutes<br />
#Add 900uL of SOC to each tube<br />
#Incubate on shaker at 37<sup>o</sup>C for 1 hour<br />
#Spread ??? uL onto plate... centrifuge first???<br />
#Incubate plates for 16 hours at 37<sup>o</sup>C. Put plates upside down to prevent condensation on surface.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:LigationWisconsin/Protocol:Ligation2007-07-05T20:52:42Z<p>Klapoetke: </p>
<hr />
<div>===Ligation===<br />
Use Nanospectrometer to find vector and insert concentration in [ng/uL]. Calculate the amount of vector and insert to use with this excel spreadsheet.</div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-07-05T20:44:14Z<p>Klapoetke: /* Protocol */</p>
<hr />
<div><center>[[Image:IGEM_soft_logo.jpg]]</center><br />
<br />
==Widgeteers==<br />
Junior Widgeteer<br />
*Nathan Klapoetke<br />
*Sean McMaster<br />
*David Peterson<br />
<br />
<br />
Chief Widgeteer<br />
*Aseem Ansari<br />
*Clay Carlson<br />
*Franco Cerrina<br />
*Mary Ozers<br />
*Doug Weibel<br />
<br />
<br />
==Project ATF==<br />
... more to come later<br />
<br />
<br />
==Protocol==<br />
<br />
===Cloning===<br />
*[[Wisconsin/Protocol:Digestion|Digestion]]<br />
*[[Wisconsin/Protocol:Ligation|Ligation]]<br />
*[[Wisconsin/Protocol:Miniprep|Miniprep]]<br />
*[[Wisconsin/Protocol:Primer|Primer Design]]<br />
*[[Wisconsin/Protocol:Transformation|Transformation]]<br />
<br />
[[Wisconsin/Protocol:Agarose Gel|Gel Electrophoresis]]<br />
<br />
[[Wisconsin/Protocol:PCR|PCR and PCR Purification]]</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:PrimerWisconsin/Protocol:Primer2007-07-04T22:08:43Z<p>Klapoetke: </p>
<hr />
<div>===Useful Websites===<br />
[http://ca.expasy.org/tools/dna.html DNA Translation]<br />
<br />
[http://www.promega.com/guides/re_guide/RESearch.asp Restriction Enzyme]<br />
<br />
[http://www.promega.com/guides/re_guide/RESearch.asp?search=buffer Restriction Enzyme Buffer]<br />
<br />
[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.asp Handles]<br />
<br />
[http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/ Oligo Analyzer]</div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-07-04T22:08:38Z<p>Klapoetke: /* Protocol */</p>
<hr />
<div><center>[[Image:IGEM_soft_logo.jpg]]</center><br />
<br />
==Widgeteers==<br />
Junior Widgeteer<br />
*Nathan Klapoetke<br />
*Sean McMaster<br />
*David Peterson<br />
<br />
<br />
Chief Widgeteer<br />
*Aseem Ansari<br />
*Clay Carlson<br />
*Franco Cerrina<br />
*Mary Ozers<br />
*Doug Weibel<br />
<br />
<br />
==Project ATF==<br />
... more to come later<br />
<br />
<br />
==Protocol==<br />
<br />
[[Wisconsin/Protocol:Agarose Gel|Agarose Gel]]<br />
<br />
[[Wisconsin/Protocol:Digestion|Digestion]]<br />
<br />
Ligation<br />
<br />
[[Wisconsin/Protocol:Miniprep|Miniprep]]<br />
<br />
[[Wisconsin/Protocol:PCR|PCR and PCR Purification]]<br />
<br />
[[Wisconsin/Protocol:Primer|Primer Design]]<br />
<br />
Transformation</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:PCRWisconsin/Protocol:PCR2007-07-04T22:08:12Z<p>Klapoetke: </p>
<hr />
<div>===Primer Stock===<br />
First check primer concentration using spectrophotometer... then prepare stock of 500uL 10uM.<br />
<br />
===Basic Non-Sequencing PCR===<br />
*38.5uL H<sub>2</sub>O<br />
*5uL 10x ExTaq buffer<br />
*1uL 10mM dNTPs<br />
*1uL Forward Primer<br />
*1uL Reverse Primer<br />
*1uL Template<br />
*0.5uL ExTaq (add this last)<br />
*2uL MgCl<sub>2</sub> 25mM<br />
<br />
Run on Ansari PCR machine CDC-02<br />
<br />
===PCR Purification===<br />
Follow instruction in the [http://www1.qiagen.com/literature/protocols/QIAquickSpin.aspx QIAquick Spin Handbook]</div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-07-04T22:08:06Z<p>Klapoetke: /* Protocol */</p>
<hr />
<div><center>[[Image:IGEM_soft_logo.jpg]]</center><br />
<br />
==Widgeteers==<br />
Junior Widgeteer<br />
*Nathan Klapoetke<br />
*Sean McMaster<br />
*David Peterson<br />
<br />
<br />
Chief Widgeteer<br />
*Aseem Ansari<br />
*Clay Carlson<br />
*Franco Cerrina<br />
*Mary Ozers<br />
*Doug Weibel<br />
<br />
<br />
==Project ATF==<br />
... more to come later<br />
<br />
<br />
==Protocol==<br />
<br />
[[Wisconsin/Protocol:Agarose Gel|Agarose Gel]]<br />
<br />
[[Wisconsin/Protocol:Digestion|Digestion]]<br />
<br />
Ligation<br />
<br />
[[Wisconsin/Protocol:Miniprep|Miniprep]]<br />
<br />
[[Wisconsin/Protocol:PCR|PCR and PCR Purification]]<br />
<br />
[[Wisconsin/Primer|Primer Design]]<br />
<br />
Transformation</div>Klapoetkehttp://2007.igem.org/wiki/index.php/Wisconsin/Protocol:MiniprepWisconsin/Protocol:Miniprep2007-07-04T22:07:10Z<p>Klapoetke: </p>
<hr />
<div>Follow instructions in [http://www.eppendorf.ca/utilities/download_pdf.asp?DR=downloads%2Fpdf%2Fmanuals%2Feppendorf&FN=FastPlasmid102504%2Epdf FastPlasmid Mini Manual].</div>Klapoetkehttp://2007.igem.org/wiki/index.php/WisconsinWisconsin2007-07-04T22:06:16Z<p>Klapoetke: /* Protocol */</p>
<hr />
<div><center>[[Image:IGEM_soft_logo.jpg]]</center><br />
<br />
==Widgeteers==<br />
Junior Widgeteer<br />
*Nathan Klapoetke<br />
*Sean McMaster<br />
*David Peterson<br />
<br />
<br />
Chief Widgeteer<br />
*Aseem Ansari<br />
*Clay Carlson<br />
*Franco Cerrina<br />
*Mary Ozers<br />
*Doug Weibel<br />
<br />
<br />
==Project ATF==<br />
... more to come later<br />
<br />
<br />
==Protocol==<br />
<br />
[[Wisconsin/Protocol:Agarose Gel|Agarose Gel]]<br />
<br />
[[Wisconsin/Protocol:Digestion|Digestion]]<br />
<br />
Ligation<br />
<br />
[[Wisconsin/Protocol:Miniprep|Miniprep]]<br />
<br />
[[Wisconsin/PCR|PCR and PCR Purification]]<br />
<br />
[[Wisconsin/Primer|Primer Design]]<br />
<br />
Transformation</div>Klapoetke