http://2009.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=100&target=Ramakrishna&year=&month=2009.igem.org - User contributions [en]2024-03-29T15:50:56ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/User:RamakrishnaUser:Ramakrishna2010-10-31T22:39:17Z<p>Ramakrishna: </p>
<hr />
<div>==Contact Info==<br />
[[Image:rama.jpg]]<br />
*Rama Krishna<br />
310, Saraswati Hostel,<br />
Indian Institute of Technology Madras<br />
Adyar <br />
Chennai 600 036<br />
Tamil Nadu<br />
India. <br />
*[[Special:Emailuser/Rama Krishna|Email me through OpenWetWare]] or ramakrishna.89@gmail.com<br />
<br />
==Education==<br />
*Undergraduate (Senior)<br />
[[Image:bt.jpg]]Department of Biotechnology, IITMadras<br />
<br />
==Research Interests==<br />
<!-- Feel free to add brief descriptions to your research interests as well --><br />
*Synthetic Biology - New ways to look at the evolution of genes and memes. <br />
I define synthetic biology as genes of one organism <br />
manipulating the genes of another with a lump of brain [[Image:madsci.jpg]]<br />
<br />
*virology - molecular dynamic simulations of proteins<br />
[[Image:prot.jpg]]<br />
<br />
==Useful links==<br />
<br />
*[http://openwetware.org/wiki/Protocols protocols]<br />
*[http://openwetware.org/wiki/Escherichia_coli E.coli]<br />
*[http://openwetware.org/wiki/IGEM:Idea_exchange Idea Exchange]<br />
'''''My Links'''''<br />
*[http://openwetware.org/wiki/Columbia_University/2009_summer/Notebook/ My 2009 summer Note Book ]<br />
*[http://openwetware.org/wiki/IGEM:Indian_Institue_of_Technology_Madras/2009/Notebook/Combinatorial_LOck My 2009 IGEM Note Book]</div>Ramakrishnahttp://2009.igem.org/User:RamakrishnaUser:Ramakrishna2010-10-31T22:38:44Z<p>Ramakrishna: /* Contact Info */</p>
<hr />
<div>==Contact Info==<br />
[[Image:rama.jpg]]<br />
*Rama Krishna<br />
310, Saraswati Hostel,<br />
Indian Institute of Technology Madras<br />
Adyar <br />
Chennai 600 036<br />
Tamil Nadu<br />
India. <br />
*[[Special:Emailuser/Rama Krishna|Email me through OpenWetWare]] or ramakrishna.89@gmail.com<br />
<br />
==Education==<br />
*Undergraduate (Senior)<br />
[[Image:bt.jpg]]Department of Biotechnology, IITMadras<br />
<br />
==Research Interests==<br />
<!-- Feel free to add brief descriptions to your research interests as well --><br />
*Synthetic Biology - New ways to look at the evolution of genes and memes. <br />
I define synthetic biology as genes of one organism <br />
manipulating the genes of another with a lump of brain [[Image:madsci.jpg]]<br />
<br />
*virology - molecular dynamic simulations of proteins<br />
[[Image:prot.jpg]]<br />
<br />
==Useful links==<br />
<br />
*[http://openwetware.org/wiki/Protocols protocols]<br />
*[http://openwetware.org/wiki/Escherichia_coli E.coli]<br />
*[http://openwetware.org/wiki/IGEM:Idea_exchange Idea Exchange]<br />
'''''My Links'''''<br />
*[http://openwetware.org/wiki/Columbia_University/2009_summer/Notebook/ My 2009 summer Note Book ]<br />
*[http://openwetware.org/wiki/IGEM:Indian_Institue_of_Technology_Madras/2009/Notebook/Combinatorial_LOck My 2009 IGEM Note Book]<br />
<br />
==Interesting Stuff==<br />
'''''papers'''''<br />
#[http://www.ncbi.nlm.nih.gov/pubmed/18414488?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum| A synthetic Escherichia coli predator prey ecosystem]</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ResultsTeam:IIT Madras/Results2009-10-22T01:50:47Z<p>Ramakrishna: /* Comparing the differences in the growth rates of cells with and without plasmids in various media */</p>
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<div>{{:Team:IITM/main}}<br />
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==Results==<br />
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=== Comparing the differences in the growth rates of cells with and without plasmids in various media ===<br />
----<br />
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<html><br><b><font color="#000">Growth curves</font></b><br></html><br />
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[[Image:clubbed.jpg|750px]]<br />
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<html><b><font color="#000">Fig 8.1: </font></b><i>a) DH5a grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics. This clearly shows that DH5a cannot grow in medium with antibiotics. b) RFP (pSB1C3) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics. c) CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics. d) RFP (pSB1C3) - CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.</i></html><br />
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[[Image:noaball4.jpg|600px]]<br />
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<html><b><font color="#000">Fig 8.2: </font></b><i>All 4 strains in media without antibiotics. This graph clearly elucidates the fact that the cells which bear a plasmid tend to grow slower than those without it. The DH5a cells show considerably faster growth rate in the exponential phase compared to all the transformed cells. We wish to use this result along with the fact that cells lose plasmids in the absence of an selection pressure to retain it, to direct plasmid loss in a very specific manner, which is the key requirement to achieve a locking system.</i></html><br />
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[[Image:corraball4.jpg|600px]]<br />
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<html><b><font color="#000">Fig 8.3: </font></b><i>All 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin.</i></html><br />
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[[Image:logall4noab.jpg|600px]]<br />
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<html><b><font color="#000">Fig 8.4: </font></b><i>log (OD600) vs Time plot for all 4 strains in media without antibiotics.</i></html><br />
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<br />
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[[Image:logcorrab.jpg|600px]]<br />
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<html><b><font color="#000">Fig 8.5: </font></b><i>log (OD600) vs Time plot for all 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin.</i></html><br />
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<html><br></html><br />
<br />
===Modeling===<br />
<br />
<html><br />
For updated results, keep checking <a href="https://2009.igem.org/User:Abdul">here.</a><br />
</html><br />
<br />
===Fluorescent Imaging===<br />
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<br />
</p><br></html><br />
[[Image:fluorescence merge pic.jpg|800px|center]]<br />
<html><p align="center"><html><b><font color="#000">Fig 9: </font></b><i>Co-transformed cells with both CFP(1A2) and RFP(1C3)seen under bright field, red filter and cyan filter.</i></html><br />
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<br />
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<br><br />
<br><br />
<br><br />
For updated results, keep checking <a href="https://2009.igem.org/User:Abdul">here.</a><br />
</html><br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ResultsTeam:IIT Madras/Results2009-10-22T01:50:02Z<p>Ramakrishna: /* Comparing the differences in the growth rates of cells with and without plasmids in various media */</p>
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==Results==<br />
<br />
=== Comparing the differences in the growth rates of cells with and without plasmids in various media ===<br />
----<br />
<br />
<html><br><b><font color="#000">Growth curves</font></b><br></html><br />
<br />
<html><br></html> <br />
[[Image:clubbed.jpg|700px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.1: </font></b><i>a) DH5a grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics. This clearly shows that DH5a cannot grow in medium with antibiotics. b) RFP (pSB1C3) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics. c) CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics. d) RFP (pSB1C3) - CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.</i></html><br />
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<br />
<br />
<html><br></html> <br />
[[Image:noaball4.jpg|600px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.2: </font></b><i>All 4 strains in media without antibiotics. This graph clearly elucidates the fact that the cells which bear a plasmid tend to grow slower than those without it. The DH5a cells show considerably faster growth rate in the exponential phase compared to all the transformed cells. We wish to use this result along with the fact that cells lose plasmids in the absence of an selection pressure to retain it, to direct plasmid loss in a very specific manner, which is the key requirement to achieve a locking system.</i></html><br />
<html><br></html> <br />
<html><br></html> <br />
<br />
<br />
<html><br></html> <br />
[[Image:corraball4.jpg|600px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.3: </font></b><i>All 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin.</i></html><br />
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<html><br></html> <br />
<br />
<br />
<html><br></html><br />
[[Image:logall4noab.jpg|600px]]<br />
<html><br></html><br />
<html><b><font color="#000">Fig 8.4: </font></b><i>log (OD600) vs Time plot for all 4 strains in media without antibiotics.</i></html><br />
<html><br></html><br />
<html><br></html><br />
<br />
<br />
<html><br></html><br />
[[Image:logcorrab.jpg|600px]]<br />
<html><br></html><br />
<html><b><font color="#000">Fig 8.5: </font></b><i>log (OD600) vs Time plot for all 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin.</i></html><br />
<html><br></html><br />
<html><br></html><br />
<br />
===Modeling===<br />
<br />
<html><br />
For updated results, keep checking <a href="https://2009.igem.org/User:Abdul">here.</a><br />
</html><br />
<br />
===Fluorescent Imaging===<br />
<html><br />
<br />
<br />
</p><br></html><br />
[[Image:fluorescence merge pic.jpg|800px|center]]<br />
<html><p align="center"><html><b><font color="#000">Fig 9: </font></b><i>Co-transformed cells with both CFP(1A2) and RFP(1C3)seen under bright field, red filter and cyan filter.</i></html><br />
<br />
<br />
<html><br />
<br />
<br><br />
<br><br />
<br><br />
For updated results, keep checking <a href="https://2009.igem.org/User:Abdul">here.</a><br />
</html><br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/File:Clubbed.jpgFile:Clubbed.jpg2009-10-22T01:46:52Z<p>Ramakrishna: </p>
<hr />
<div></div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ResultsTeam:IIT Madras/Results2009-10-22T01:46:10Z<p>Ramakrishna: /* Comparing the differences in the growth rates of cells with and without plasmids in various media */</p>
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<div>{{:Team:IITM/main}}<br />
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==Results==<br />
<br />
=== Comparing the differences in the growth rates of cells with and without plasmids in various media ===<br />
----<br />
<br />
<html><br><b><font color="#000">Growth curves</font></b><br></html><br />
<br />
<html><br></html> <br />
[[Image:clubbed.jpg|600px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.1: </font></b><i>a) DH5a grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics. This clearly shows that DH5a cannot grow in medium with antibiotics. b) RFP (pSB1C3) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics. c) CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics. d) RFP (pSB1C3) - CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.</i></html><br />
<html><br></html> <br />
<html><br></html> <br />
<br />
<br />
<html><br></html> <br />
[[Image:noaball4.jpg|600px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.2: </font></b><i>All 4 strains in media without antibiotics. This graph clearly elucidates the fact that the cells which bear a plasmid tend to grow slower than those without it. The DH5a cells show considerably faster growth rate in the exponential phase compared to all the transformed cells. We wish to use this result along with the fact that cells lose plasmids in the absence of an selection pressure to retain it, to direct plasmid loss in a very specific manner, which is the key requirement to achieve a locking system.</i></html><br />
<html><br></html> <br />
<html><br></html> <br />
<br />
<br />
<html><br></html> <br />
[[Image:corraball4.jpg|600px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.3: </font></b><i>All 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin.</i></html><br />
<html><br></html> <br />
<html><br></html> <br />
<br />
<br />
<html><br></html><br />
[[Image:logall4noab.jpg|600px]]<br />
<html><br></html><br />
<html><b><font color="#000">Fig 8.4: </font></b><i>log (OD600) vs Time plot for all 4 strains in media without antibiotics.</i></html><br />
<html><br></html><br />
<html><br></html><br />
<br />
<br />
<html><br></html><br />
[[Image:logcorrab.jpg|600px]]<br />
<html><br></html><br />
<html><b><font color="#000">Fig 8.5: </font></b><i>log (OD600) vs Time plot for all 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin.</i></html><br />
<html><br></html><br />
<html><br></html><br />
<br />
===Modeling===<br />
<br />
<html><br />
For updated results, keep checking <a href="https://2009.igem.org/User:Abdul">here.</a><br />
</html><br />
<br />
===Fluorescent Imaging===<br />
<html><br />
<br />
<br />
</p><br></html><br />
[[Image:fluorescence merge pic.jpg|800px|center]]<br />
<html><p align="center"><html><b><font color="#000">Fig 9: </font></b><i>Co-transformed cells with both CFP(1A2) and RFP(1C3)seen under bright field, red filter and cyan filter.</i></html><br />
<br />
<br />
<html><br />
<br />
<br><br />
<br><br />
<br><br />
For updated results, keep checking <a href="https://2009.igem.org/User:Abdul">here.</a><br />
</html><br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/Notebook/Parts_SubmittedTeam:IIT Madras/Notebook/Parts Submitted2009-10-22T01:31:29Z<p>Ramakrishna: </p>
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<div>{{:Team:IITM/main}}<br />
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<p> We have submitted 2 parts to the Registry of Standard Biological Parts.</p><br />
<br />
<ul type="disc"><br />
<li><b><font color="#000">K272001</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for RFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/d/d2/K272001.jpg" align="middle" width="300" height="60"></a> <br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>607</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>584</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>mRFP1</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272001">here</a> to access K272001 in Registry.</p><br />
<br />
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[[Image:rfpcells.jpg|600px]]<br />
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Fig 1: A crude image of cells transformed with K272001 through various filters. a) Cells in bright field b) Cells under a Cyan fluorescence filter (do not show any fluorescence) c) Cells under a Red fluorescence filter<br />
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<li><b><font color="#000">K272002</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for CFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/8/87/K272002.jpg" align="left" width="300" height="60"></a><br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>476</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>439</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>ECFP</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272002">here</a> to access K272002 in Registry.</p><br />
<html><br></html><br />
[[Image:cfpcells.jpg|600px]]<br />
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Fig 2: An image of cells transformed with K272002 through various filters. a) Cells in bright field b) Cells under a Cyan fluorescence filter c) Cells under a Red fluorescence filter (do not show any fluorescence) <br />
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{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/Notebook/SandboxTeam:IIT Madras/Notebook/Sandbox2009-10-22T01:30:26Z<p>Ramakrishna: /* Intermediates planned through PCR protocol */</p>
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padding: 10px 10px 60px 10px;<br />
width: 800px;<br />
color: #000;<br />
}<br />
<br />
</style><br />
<br />
<STYLE TYPE="text/css"><br />
<!--<br />
.justifyalign {text-align:justify}<br />
--><br />
</STYLE><br />
<br />
</head><br />
<br />
<body><br />
<br />
<div id="aContent"><br />
</html><br />
<br />
==Intermediates and proposed constructs for the the 2 plasmid locking system==<br />
<br />
==Original design==<br />
<br />
In this design, each of the construct has its own antibiotic resistance marker (independent of the plasmid backbone marker).<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Plasmid 1</font></b><br />
<br />
<html><br></html><br />
<br />
[[Image:2plasmidoriginaldesignplasmid1.jpg]]<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Plasmid 2</font></b><br />
<br />
<html><br></html><br />
<br />
[[Image:2plasmidoriginaldesignplasmid2.jpg]]<br />
<br />
<html><br></html><br />
<br />
==Final design==<br />
<br />
We decided to remove the extra antibiotic markers from the inserts. Then it looks like this<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Plasmid 1</font></b><br />
<br />
<html><br></html><br />
<br />
We had more than one way to build plasmid 1<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Path 1</font></b><br />
<br />
[[Image:2plasmidfinaldesignplasmid1path1.jpg]]<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Path 1</font></b><br />
<br />
[[Image:2plasmidfinaldesignplasmid1path2.jpg]]<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Plasmid 2</font></b><br />
<br />
<html><br></html><br />
<br />
[[Image:2plasmidfinaldesignplasmid2.jpg]]<br />
<br />
<html><br></html><br />
<br />
==Intermediates and proposed constructs for the the 3 plasmid locking system==<br />
<br />
We have designed the constructs for the 3 plasmid system, but we haven't planned to build it yet. Some of the parts built for the 2 plasmid system could be reused for the 3 plasmid system too.<br />
<br />
== Intermediates planned through PCR protocol ==<br />
<br />
<html><br></html><br />
<br />
These parts are essentially same as the those that occur in the construction strategies that are shown above.<br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/File:Cfpcells.jpgFile:Cfpcells.jpg2009-10-22T01:29:13Z<p>Ramakrishna: </p>
<hr />
<div></div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/Notebook/SandboxTeam:IIT Madras/Notebook/Sandbox2009-10-22T01:20:53Z<p>Ramakrishna: /* Intermediates planned through PCR protocol */</p>
<hr />
<div>{{:Team:IITM/main}}<br />
<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br />
<head><style><br />
<br />
body<br />
{<br />
background: #000;<br />
}<br />
<br />
#aContent<br />
{<br />
background-color:#fff;<br />
margin: 50px auto;<br />
padding: 10px 10px 60px 10px;<br />
width: 800px;<br />
color: #000;<br />
}<br />
<br />
</style><br />
<br />
<STYLE TYPE="text/css"><br />
<!--<br />
.justifyalign {text-align:justify}<br />
--><br />
</STYLE><br />
<br />
</head><br />
<br />
<body><br />
<br />
<div id="aContent"><br />
</html><br />
<br />
==Intermediates and proposed constructs for the the 2 plasmid locking system==<br />
<br />
==Original design==<br />
<br />
In this design, each of the construct has its own antibiotic resistance marker (independent of the plasmid backbone marker).<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Plasmid 1</font></b><br />
<br />
<html><br></html><br />
<br />
[[Image:2plasmidoriginaldesignplasmid1.jpg]]<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Plasmid 2</font></b><br />
<br />
<html><br></html><br />
<br />
[[Image:2plasmidoriginaldesignplasmid2.jpg]]<br />
<br />
<html><br></html><br />
<br />
==Final design==<br />
<br />
We decided to remove the extra antibiotic markers from the inserts. Then it looks like this<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Plasmid 1</font></b><br />
<br />
<html><br></html><br />
<br />
We had more than one way to build plasmid 1<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Path 1</font></b><br />
<br />
[[Image:2plasmidfinaldesignplasmid1path1.jpg]]<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Path 1</font></b><br />
<br />
[[Image:2plasmidfinaldesignplasmid1path2.jpg]]<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Plasmid 2</font></b><br />
<br />
<html><br></html><br />
<br />
[[Image:2plasmidfinaldesignplasmid2.jpg]]<br />
<br />
<html><br></html><br />
<br />
==Intermediates and proposed constructs for the the 3 plasmid locking system==<br />
<br />
We have designed the constructs for the 3 plasmid system, but we haven't planned to build it yet. Some of the parts built for the 2 plasmid system could be reused for the 3 plasmid system too.<br />
<br />
== Intermediates planned through PCR protocol ==<br />
<br />
<html><br></html><br />
<br />
These parts are essentially same as the those that occur in the construction strategies that are shown above.<br />
[[Image:cfpcells.jpg]]<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/Notebook/Parts_SubmittedTeam:IIT Madras/Notebook/Parts Submitted2009-10-22T01:20:15Z<p>Ramakrishna: </p>
<hr />
<div>{{:Team:IITM/main}}<br />
<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<head><style><br />
<br />
body<br />
{<br />
background: #000;<br />
}<br />
<br />
#aContent<br />
{<br />
background-color:#fff;<br />
margin: 50px auto;<br />
padding: 10px 10px 60px 10px;<br />
width: 800px;<br />
color: #000;<br />
}<br />
<br />
</style><br />
<br />
<STYLE TYPE="text/css"><br />
<!--<br />
.justifyalign {text-align:justify}<br />
--><br />
</STYLE><br />
<br />
</head><br />
<br />
<body><br />
<br />
<div id="aContent"><br />
<br />
<p> We have submitted 2 parts to the Registry of Standard Biological Parts.</p><br />
<br />
<ul type="disc"><br />
<li><b><font color="#000">K272001</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for RFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/d/d2/K272001.jpg" align="middle" width="300" height="60"></a> <br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>607</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>584</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>mRFP1</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272001">here</a> to access K272001 in Registry.</p><br />
<br />
<html><br></html><br />
[[Image:rfpcells.jpg|600px]]<br />
<html><br></html><br />
Fig 1: A crude image of cells transformed with K272001 through various filters. a) Cells in bright field b) Cells under a Cyan fluorescence filter (do not show any fluorescence) c) Cells under a Red fluorescence filter<br />
<br />
<html><br />
<br><br><br><br />
<br />
<li><b><font color="#000">K272002</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for CFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/8/87/K272002.jpg" align="left" width="300" height="60"></a><br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>476</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>439</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>ECFP</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272002">here</a> to access K272002 in Registry.</p><br />
</ul><br />
</div><br />
</body><br />
</html><br />
<br />
[[Image:cfpcells.jpg|600px]]<br />
<html><br></html><br />
Fig 1: A crude image of cells transformed with K272002 through various filters. a) Cells in bright field b) Cells under a Cyan fluorescence filter c) Cells under a Red fluorescence filter (do not show any fluorescence) <br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/Notebook/Parts_SubmittedTeam:IIT Madras/Notebook/Parts Submitted2009-10-22T01:19:23Z<p>Ramakrishna: </p>
<hr />
<div>{{:Team:IITM/main}}<br />
<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<head><style><br />
<br />
body<br />
{<br />
background: #000;<br />
}<br />
<br />
#aContent<br />
{<br />
background-color:#fff;<br />
margin: 50px auto;<br />
padding: 10px 10px 60px 10px;<br />
width: 800px;<br />
color: #000;<br />
}<br />
<br />
</style><br />
<br />
<STYLE TYPE="text/css"><br />
<!--<br />
.justifyalign {text-align:justify}<br />
--><br />
</STYLE><br />
<br />
</head><br />
<br />
<body><br />
<br />
<div id="aContent"><br />
<br />
<p> We have submitted 2 parts to the Registry of Standard Biological Parts.</p><br />
<br />
<ul type="disc"><br />
<li><b><font color="#000">K272001</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for RFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/d/d2/K272001.jpg" align="middle" width="300" height="60"></a> <br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>607</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>584</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>mRFP1</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272001">here</a> to access K272001 in Registry.</p><br />
<br />
<html><br></html><br />
[[Image:rfpcells.jpg|600px]]<br />
Fig 1: A crude image of cells transformed with K272001 through various filters. a) Cells in bright field b) Cells under a Cyan fluorescence filter (do not show any fluorescence) c) Cells under a Red fluorescence filter<br />
<br />
<html><br />
<br><br><br><br />
<br />
<li><b><font color="#000">K272002</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for CFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/8/87/K272002.jpg" align="left" width="300" height="60"></a><br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>476</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>439</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>ECFP</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272002">here</a> to access K272002 in Registry.</p><br />
</ul><br />
</div><br />
</body><br />
</html><br />
<br />
[[Image:rfpcells.jpg|600px]]<br />
Fig 1: A crude image of cells transformed with K272002 through various filters. a) Cells in bright field b) Cells under a Cyan fluorescence filter c) Cells under a Red fluorescence filter (do not show any fluorescence) <br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/Notebook/Parts_SubmittedTeam:IIT Madras/Notebook/Parts Submitted2009-10-22T01:16:54Z<p>Ramakrishna: </p>
<hr />
<div>{{:Team:IITM/main}}<br />
<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<head><style><br />
<br />
body<br />
{<br />
background: #000;<br />
}<br />
<br />
#aContent<br />
{<br />
background-color:#fff;<br />
margin: 50px auto;<br />
padding: 10px 10px 60px 10px;<br />
width: 800px;<br />
color: #000;<br />
}<br />
<br />
</style><br />
<br />
<STYLE TYPE="text/css"><br />
<!--<br />
.justifyalign {text-align:justify}<br />
--><br />
</STYLE><br />
<br />
</head><br />
<br />
<body><br />
<br />
<div id="aContent"><br />
<br />
<p> We have submitted 2 parts to the Registry of Standard Biological Parts.</p><br />
<br />
<ul type="disc"><br />
<li><b><font color="#000">K272001</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for RFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/d/d2/K272001.jpg" align="middle" width="300" height="60"></a> <br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>607</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>584</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>mRFP1</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272001">here</a> to access K272001 in Registry.</p><br />
<br />
<html><br></html><br />
[[Image:rfpcells.jpg]]<br />
Fig 1: A crude image of cells with various filters. a) Cells in bright field b) Cells under a Cyan fluorescence filter (does not show any fluorescence) c) Cells under a Red fluorescence filter<br />
<br />
<html><br />
<br><br><br><br />
<br />
<li><b><font color="#000">K272002</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for CFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/8/87/K272002.jpg" align="left" width="300" height="60"></a><br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>476</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>439</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>ECFP</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272002">here</a> to access K272002 in Registry.</p><br />
</ul><br />
</div><br />
</body><br />
</html><br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/File:Rfpcells.jpgFile:Rfpcells.jpg2009-10-22T01:14:45Z<p>Ramakrishna: </p>
<hr />
<div></div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/Notebook/SandboxTeam:IIT Madras/Notebook/Sandbox2009-10-22T01:14:15Z<p>Ramakrishna: /* Intermediates planned through PCR protocol */</p>
<hr />
<div>{{:Team:IITM/main}}<br />
<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br><br><br />
<head><style><br />
<br />
body<br />
{<br />
background: #000;<br />
}<br />
<br />
#aContent<br />
{<br />
background-color:#fff;<br />
margin: 50px auto;<br />
padding: 10px 10px 60px 10px;<br />
width: 800px;<br />
color: #000;<br />
}<br />
<br />
</style><br />
<br />
<STYLE TYPE="text/css"><br />
<!--<br />
.justifyalign {text-align:justify}<br />
--><br />
</STYLE><br />
<br />
</head><br />
<br />
<body><br />
<br />
<div id="aContent"><br />
</html><br />
<br />
==Intermediates and proposed constructs for the the 2 plasmid locking system==<br />
<br />
==Original design==<br />
<br />
In this design, each of the construct has its own antibiotic resistance marker (independent of the plasmid backbone marker).<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Plasmid 1</font></b><br />
<br />
<html><br></html><br />
<br />
[[Image:2plasmidoriginaldesignplasmid1.jpg]]<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Plasmid 2</font></b><br />
<br />
<html><br></html><br />
<br />
[[Image:2plasmidoriginaldesignplasmid2.jpg]]<br />
<br />
<html><br></html><br />
<br />
==Final design==<br />
<br />
We decided to remove the extra antibiotic markers from the inserts. Then it looks like this<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Plasmid 1</font></b><br />
<br />
<html><br></html><br />
<br />
We had more than one way to build plasmid 1<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Path 1</font></b><br />
<br />
[[Image:2plasmidfinaldesignplasmid1path1.jpg]]<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Path 1</font></b><br />
<br />
[[Image:2plasmidfinaldesignplasmid1path2.jpg]]<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Plasmid 2</font></b><br />
<br />
<html><br></html><br />
<br />
[[Image:2plasmidfinaldesignplasmid2.jpg]]<br />
<br />
<html><br></html><br />
<br />
==Intermediates and proposed constructs for the the 3 plasmid locking system==<br />
<br />
We have designed the constructs for the 3 plasmid system, but we haven't planned to build it yet. Some of the parts built for the 2 plasmid system could be reused for the 3 plasmid system too.<br />
<br />
== Intermediates planned through PCR protocol ==<br />
<br />
<html><br></html><br />
<br />
These parts are essentially same as the those that occur in the construction strategies that are shown above.<br />
[[Image:rfpcells.jpg]]<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/Notebook/Parts_SubmittedTeam:IIT Madras/Notebook/Parts Submitted2009-10-22T01:11:29Z<p>Ramakrishna: </p>
<hr />
<div>{{:Team:IITM/main}}<br />
<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<head><style><br />
<br />
body<br />
{<br />
background: #000;<br />
}<br />
<br />
#aContent<br />
{<br />
background-color:#fff;<br />
margin: 50px auto;<br />
padding: 10px 10px 60px 10px;<br />
width: 800px;<br />
color: #000;<br />
}<br />
<br />
</style><br />
<br />
<STYLE TYPE="text/css"><br />
<!--<br />
.justifyalign {text-align:justify}<br />
--><br />
</STYLE><br />
<br />
</head><br />
<br />
<body><br />
<br />
<div id="aContent"><br />
<br />
<p> We have submitted 2 parts to the Registry of Standard Biological Parts.</p><br />
<br />
<ul type="disc"><br />
<li><b><font color="#000">K272001</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for RFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/d/d2/K272001.jpg" align="middle" width="300" height="60"></a> <br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>607</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>584</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>mRFP1</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272001">here</a> to access K272001 in Registry.</p><br />
<br />
<br />
[[Image:rfpcells.jpg]]<br />
Fig 1: A crude image of cells with various filters. a) Cells in bright field b) Cells under a Cyan fluorescence filter (does not show any fluorescence) c) Cells under a Red fluorescence filter<br />
<br />
<br />
<br><br><br><br />
<br />
<li><b><font color="#000">K272002</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for CFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/8/87/K272002.jpg" align="left" width="300" height="60"></a><br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>476</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>439</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>ECFP</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272002">here</a> to access K272002 in Registry.</p><br />
</ul><br />
</div><br />
</body><br />
</html><br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/Notebook/Parts_SubmittedTeam:IIT Madras/Notebook/Parts Submitted2009-10-22T01:11:02Z<p>Ramakrishna: </p>
<hr />
<div>{{:Team:IITM/main}}<br />
<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<head><style><br />
<br />
body<br />
{<br />
background: #000;<br />
}<br />
<br />
#aContent<br />
{<br />
background-color:#fff;<br />
margin: 50px auto;<br />
padding: 10px 10px 60px 10px;<br />
width: 800px;<br />
color: #000;<br />
}<br />
<br />
</style><br />
<br />
<STYLE TYPE="text/css"><br />
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.justifyalign {text-align:justify}<br />
--><br />
</STYLE><br />
<br />
</head><br />
<br />
<body><br />
<br />
<div id="aContent"><br />
<br />
<p> We have submitted 2 parts to the Registry of Standard Biological Parts.</p><br />
<br />
<ul type="disc"><br />
<li><b><font color="#000">K272001</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for RFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/d/d2/K272001.jpg" align="middle" width="300" height="60"></a> <br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>607</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>584</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>mRFP1</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272001">here</a> to access K272001 in Registry.</p><br />
<br />
<html><br></html><br />
[[Image:rfpcells.jpg]]<br />
Fig 1: A crude image of cells with various filters. a) Cells in bright field b) Cells under a Cyan fluorescence filter (does not show any fluorescence) c) Cells under a Red fluorescence filter<br />
<br />
<br />
<br><br><br><br />
<br />
<li><b><font color="#000">K272002</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for CFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/8/87/K272002.jpg" align="left" width="300" height="60"></a><br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>476</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>439</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>ECFP</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272002">here</a> to access K272002 in Registry.</p><br />
</ul><br />
</div><br />
</body><br />
</html><br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/Notebook/Parts_SubmittedTeam:IIT Madras/Notebook/Parts Submitted2009-10-22T01:10:25Z<p>Ramakrishna: </p>
<hr />
<div>{{:Team:IITM/main}}<br />
<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<head><style><br />
<br />
body<br />
{<br />
background: #000;<br />
}<br />
<br />
#aContent<br />
{<br />
background-color:#fff;<br />
margin: 50px auto;<br />
padding: 10px 10px 60px 10px;<br />
width: 800px;<br />
color: #000;<br />
}<br />
<br />
</style><br />
<br />
<STYLE TYPE="text/css"><br />
<!--<br />
.justifyalign {text-align:justify}<br />
--><br />
</STYLE><br />
<br />
</head><br />
<br />
<body><br />
<br />
<div id="aContent"><br />
<br />
<p> We have submitted 2 parts to the Registry of Standard Biological Parts.</p><br />
<br />
<ul type="disc"><br />
<li><b><font color="#000">K272001</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for RFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/d/d2/K272001.jpg" align="middle" width="300" height="60"></a> <br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>607</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>584</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>mRFP1</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272001">here</a> to access K272001 in Registry.</p><br />
<br />
<html><br></html><br />
[[Image:rfpcells.jpg]]<br />
Fig 1: A crude image of cells with various filters. a) Cells in bright field b) Cells under a Cyan fluorescence filter (does not show any fluorescence) c) Cells under a Red fluorescence filter<br />
<html><br></html><br />
<br />
<br><br><br><br />
<br />
<li><b><font color="#000">K272002</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for CFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/8/87/K272002.jpg" align="left" width="300" height="60"></a><br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>476</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>439</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>ECFP</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272002">here</a> to access K272002 in Registry.</p><br />
</ul><br />
</div><br />
</body><br />
</html><br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/Notebook/Parts_SubmittedTeam:IIT Madras/Notebook/Parts Submitted2009-10-22T01:08:56Z<p>Ramakrishna: </p>
<hr />
<div>{{:Team:IITM/main}}<br />
<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<head><style><br />
<br />
body<br />
{<br />
background: #000;<br />
}<br />
<br />
#aContent<br />
{<br />
background-color:#fff;<br />
margin: 50px auto;<br />
padding: 10px 10px 60px 10px;<br />
width: 800px;<br />
color: #000;<br />
}<br />
<br />
</style><br />
<br />
<STYLE TYPE="text/css"><br />
<!--<br />
.justifyalign {text-align:justify}<br />
--><br />
</STYLE><br />
<br />
</head><br />
<br />
<body><br />
<br />
<div id="aContent"><br />
<br />
<p> We have submitted 2 parts to the Registry of Standard Biological Parts.</p><br />
<br />
<ul type="disc"><br />
<li><b><font color="#000">K272001</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for RFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/d/d2/K272001.jpg" align="middle" width="300" height="60"></a> <br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>607</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>584</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>mRFP1</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<html><br></html><br />
[[Image:rfpcells.jpg]]<br />
Fig 1: A crude image of cells with various filters. a) Cells in bright field b) Cells under a Cyan fluorescence filter (does not show any fluorescence) c) Cells under a Red fluorescence filter<br />
<html><br></html><br />
<br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272001">here</a> to access K272001 in Registry.</p><br />
<br />
<br><br><br><br />
<br />
<li><b><font color="#000">K272002</font></b></li><br />
<br><br />
<b><font color="#000">Constitutive Expression Cassette for CFP</font></b><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2009/8/87/K272002.jpg" align="left" width="300" height="60"></a><br />
<br><br><br />
<br />
<p><b><font color="#000">Parameters</font></b><br />
<br><br />
<br />
<table border="1"><br />
<tr><br />
<td>Emit</td><br />
<td>476</td><br />
</tr><tr><br />
<td>Excite</td><br />
<td>439</td><br />
</tr><tr><br />
<td>Protein</td><br />
<td>ECFP</td><br />
</tr><tr><br />
<td>Tag</td><br />
<td>None</td><br />
</table><br />
<br />
<br><br />
<p>Click <a href="http://partsregistry.org/Part:BBa_K272002">here</a> to access K272002 in Registry.</p><br />
</ul><br />
</div><br />
</body><br />
</html><br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ResultsTeam:IIT Madras/Results2009-10-22T00:46:55Z<p>Ramakrishna: </p>
<hr />
<div>{{:Team:IITM/main}}<br />
<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<head><style><br />
<br />
body<br />
{<br />
background: #000;<br />
}<br />
<br />
#aContent<br />
{<br />
background-color:#fff;<br />
margin: 50px auto;<br />
padding: 10px 10px 60px 10px;<br />
width: 800px;<br />
color: #000;<br />
}<br />
<br />
</style><br />
<br />
<STYLE TYPE="text/css"><br />
<!--<br />
.justifyalign {text-align:justify}<br />
--><br />
</STYLE><br />
<br />
</head><br />
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<body><br />
<br />
<br />
<div id="aContent"><br />
</html><br />
==Results==<br />
<br />
=== Comparing the differences in the growth rates of cells with and without plasmids in various media ===<br />
----<br />
<br />
<html><br><b><font color="#000">Growth curves</font></b><br></html><br />
<br />
<html><br></html> <br />
[[Image:DH5aall4cases.jpg|600px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.1: </font></b><i>a) DH5a grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics. This clearly shows that DH5a cannot grow in medium with antibiotics. b) RFP (pSB1C3) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics. c) CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics. d) RFP (pSB1C3) - CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.</i></html><br />
<html><br></html> <br />
<html><br></html> <br />
<br />
<br />
<html><br></html> <br />
[[Image:noaball4.jpg|600px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.2: </font></b><i>All 4 strains in media without antibiotics. This graph clearly elucidates the fact that the cells which bear a plasmid tend to grow slower than those without it. The DH5a cells show considerably faster growth rate in the exponential phase compared to all the transformed cells. We wish to use this result along with the fact that cells lose plasmids in the absence of an selection pressure to retain it, to direct plasmid loss in a very specific manner, which is the key requirement to achieve a locking system.</i></html><br />
<html><br></html> <br />
<html><br></html> <br />
<br />
<br />
<html><br></html> <br />
[[Image:corraball4.jpg|600px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.3: </font></b><i>All 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin.</i></html><br />
<html><br></html> <br />
<html><br></html> <br />
<br />
<br />
<html><br></html><br />
[[Image:logall4noab.jpg|600px]]<br />
<html><br></html><br />
<html><b><font color="#000">Fig 8.4: </font></b><i>log (OD600) vs Time plot for all 4 strains in media without antibiotics.</i></html><br />
<html><br></html><br />
<html><br></html><br />
<br />
<br />
<html><br></html><br />
[[Image:logcorrab.jpg|600px]]<br />
<html><br></html><br />
<html><b><font color="#000">Fig 8.5: </font></b><i>log (OD600) vs Time plot for all 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin.</i></html><br />
<html><br></html><br />
<html><br></html><br />
<br />
===Modeling===<br />
<br />
<html><br />
For updated results, keep checking <a href="https://2009.igem.org/User:Abdul">here.</a><br />
</html><br />
<br />
===Fluorescent Imaging===<br />
<html><br />
<br />
<p>The panel below shows cells co-transformed with both constructs, namely expressing CFP with Ampicillin and RFP with Chloramphenicol.<br><br><br />
</p><br></html><br />
[[Image:brightfield.jpg|center]]<br />
<html><p align="center">(a) shows cells exposed to Bright Field.</p><br></html><br />
[[Image:RFP.jpg|center]]<br />
<html><p align="center">(b) shows cells exposed to Red filter.</p><br></html><br />
[[Image:CFP.jpg|center]]<br />
<html><p align="center">(c) shows cells exposed to Cyan filter.</p><br></html><br />
<br />
<html><br />
<br />
<br><br />
<br><br />
<br><br />
For updated results, keep checking <a href="https://2009.igem.org/User:Abdul">here.</a><br />
</html><br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ResultsTeam:IIT Madras/Results2009-10-22T00:31:35Z<p>Ramakrishna: </p>
<hr />
<div>{{:Team:IITM/main}}<br />
<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<head><style><br />
<br />
body<br />
{<br />
background: #000;<br />
}<br />
<br />
#aContent<br />
{<br />
background-color:#fff;<br />
margin: 50px auto;<br />
padding: 10px 10px 60px 10px;<br />
width: 800px;<br />
color: #000;<br />
}<br />
<br />
</style><br />
<br />
<STYLE TYPE="text/css"><br />
<!--<br />
.justifyalign {text-align:justify}<br />
--><br />
</STYLE><br />
<br />
</head><br />
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<body><br />
<br />
<br />
<div id="aContent"><br />
</html><br />
==Results==<br />
<br />
=== Comparing the differences in the growth rates of cells with and without plasmids in various media ===<br />
----<br />
<br />
<html><br><b><font color="#000">Growth curves</font></b><br></html><br />
<br />
<html><br></html> <br />
[[Image:DH5aall4cases.jpg|600px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.1: </font></b><i>DH5a grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics. This clearly shows that DH5a cannot grow in medium with antibiotics.</i></html><br />
<html><br></html> <br />
<html><br></html> <br />
<br />
<html><br></html> <br />
[[Image:rfpall4cases.jpg|600px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.2: </font></b><i>RFP (pSB1C3) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.</i></html><br />
<html><br></html> <br />
<html><br></html> <br />
<br />
<html><br></html> <br />
[[Image:cfpall4cases.jpg|600px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.3: </font></b><i>CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.</i></html><br />
<html><br></html> <br />
<html><br></html> <br />
<br />
<br />
<html><br></html> <br />
[[Image:RC4cases.jpg|600px]]<br />
<html><br></html><br />
<html><b><font color="#000">Fig 8.4: </font></b><i>RFP (pSB1C3) - CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.</i></html><br />
<html><br></html><br />
<html><br></html><br />
<br />
<br />
<html><br></html> <br />
[[Image:noaball4.jpg|600px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.5: </font></b><i>All 4 strains in media without antibiotics. This graph clearly elucidates the fact that the cells which bear a plasmid tend to grow slower than those without it. The DH5a cells show considerably faster growth rate in the exponential phase compared to all the transformed cells. </i></html><br />
<html><br></html> <br />
<html><br></html> <br />
<br />
<br />
<html><br></html> <br />
[[Image:corraball4.jpg|600px]]<br />
<html><br></html> <br />
<html><b><font color="#000">Fig 8.6: </font></b><i>All 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin.</i></html><br />
<html><br></html> <br />
<html><br></html> <br />
<br />
<br />
<html><br></html><br />
[[Image:logall4noab.jpg|600px]]<br />
<html><br></html><br />
<html><b><font color="#000">Fig 8.7: </font></b><i>log (OD600) vs Time plot for all 4 strains in media without antibiotics.</i></html><br />
<html><br></html><br />
<html><br></html><br />
<br />
<br />
<html><br></html><br />
[[Image:logcorrab.jpg|600px]]<br />
<html><br></html><br />
<html><b><font color="#000">Fig 8.8: </font></b><i>log (OD600) vs Time plot for all 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin.</i></html><br />
<html><br></html><br />
<html><br></html><br />
<br />
===Modeling===<br />
<br />
<html><br />
For updated results, keep checking <a href="https://2009.igem.org/User:Abdul">here.</a><br />
</html><br />
<br />
===Fluorescent Imaging===<br />
<html><br />
<br />
<p>The panel below shows cells co-transformed with both constructs, namely expressing CFP with Ampicillin and RFP with Chloramphenicol.<br><br><br />
</p><br></html><br />
[[Image:brightfield.jpg|center]]<br />
<html><p align="center">(a) shows cells exposed to Bright Field.</p><br></html><br />
[[Image:RFP.jpg|center]]<br />
<html><p align="center">(b) shows cells exposed to Red filter.</p><br></html><br />
[[Image:CFP.jpg|center]]<br />
<html><p align="center">(c) shows cells exposed to Cyan filter.</p><br></html><br />
<br />
<html><br />
<br />
<br><br />
<br><br />
<br><br />
For updated results, keep checking <a href="https://2009.igem.org/User:Abdul">here.</a><br />
</html><br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/Team_underTeam:IIT Madras/Team under2009-10-22T00:21:40Z<p>Ramakrishna: </p>
<hr />
<div>{{:Team:IITM/main}}<br />
<br />
<html><br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<head><style><br />
<br />
body<br />
{<br />
background: #000;<br />
}<br />
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<b><font color="#000">Abdul Majeed</font></b><br />
<p CLASS="justifyalign">I am currently in my fourth year, pursuing a dual degree in Bioengineering at IIT Madras. I did my schooling at Dubai and then came to India for professional studies. Well, my research interests include Systems Biology and Biochemical engineering. But since the past 8 months while working for iGEM 2009, I developed a passion for Synthetic Biology. My past research experience include working for Dr.Reed's group on Constrain based network analysis of sugar fermentation in <i>Saccharomyces Cerevisiae</i> at University of Wisconsin, Madison(Summer 2009) and an Industrial experience at Dr. Reddys Laboratory(Summer 2008). I enjoy playing and watching soccer. I aim high in life and try to keep up to my set standards. See you guys at the Jamboree!</p><br />
Email:<a href="mailto:m.abdul.majeed1@gmail.com">m.abdul.majeed1@gmail.com</a><br><br />
Click <a href="http://openwetware.org/wiki/User:Mohammed_Abdul_Majeed"><font color="0000FF">here</font></a> for my webpage.<br />
</td><br />
<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/3/31/Abdul1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Ajit Kamath</font></b><br />
<p CLASS="justifyalign">I am from Mumbai, India. I love playing with cells and making them do our bidding. Apart from synthetic biology, I am also interested in Developmental Immunology. I was in Bonn, Germany for the last summer studying Eosinophil activation and development in mice.</p> <br />
<p CLASS="justifyalign">I shall finish my Masters from IIT Madras in 2011. Apart from playing with cells, I like playing with computers. Some of my hobbies include cooking, reading, writing blogs and music. </p><br />
<p CLASS="justifyalign">My Biggest and Dearest Hobby: Cracking the vaguest of Jokes. Ask my Team mates for confirmation. ;)</p><br />
<p CLASS="justifyalign">To hear some of them buzz me at <a href="mailto:ajit9988@gmail.com">ajit9988@gmail.com</a></p><br />
<br><br />
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<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/1/19/Ajit1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Arun Murali</font></b><br />
<p CLASS="justifyalign">Hello World. Currently in my fourth year in the Department of Biotechnology, I am primarily interested in Molecular Biology. As for my research experience, I was in Germany this summer working on certain cell death pathways and interaction studies between proteins involved in the pathways.<br><br />
<br />
Coming to my hobbies and other eccentricities, I play Football and Table Tennis, participate in a range of Literary activities (quizzes, Creative writing, Dramatics, word games etc), and also listen to most kinds of music, though I prefer alternative metal. I also have a vague fascination with the number 19. Oh and the Flying Spaghetti Monster…. His Noodliness Rocks!<br><br />
<br />
P.S. Chuck Norris has ATP, only for him it is Adenosine Tetra Phosphate! (One of those lab nights where we were waiting for a gel to run, right Srivats?)</p><br />
Email:<a href="mailto:arun19murali@gmail.com">arun19murali@gmail.com</a><br><br />
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<img src="https://static.igem.org/mediawiki/2009/5/55/Arun1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Bhavya B</font></b><br />
<p CLASS="justifyalign">Hi! Iam currently in fourth year, pursuing a Dual Degree from the Dept of Biotechnology, IIT Madras.I had a great time working for iGEM 2009. It helped me in gaining knowledge about synthetic biology,which adds the spirit of engineering to biology.Working in labs was never more fun and I am gonna miss all the lab hours and the night outs!<br><br />
As for my research experience, I spent this summer(May-July 2009) working as an intern in BIOCON (India) in the cell culture lab where i studied the amino acid consumption patterns of mammalian (CHO) cells.<br><br />
Academics aside, i love music, dancing, spending lazy days with food and fictions, trying my hand at cooking and most of all shopping!!Looking forward to see you all at the Jamboree! </p><br />
Email:<a href="mailto:brundavanambhavya@gmail.com">brundavanambhavya@gmail.com</a><br><br />
</td><br />
<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/2/2c/Bhavya1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Harshvardhan</font></b><br />
<p CLASS="justifyalign">Namaste everyone,<br />
This is Harsh a.k.a takla, yogi, bokka etc. I dont like boasting about myself, because i am so intelligent and smart that I don't need to talk about it ;) Just kidding, but seriously ;) ;) <br><br />
I feel lucky to be in the iGEM'09 team of IITMadras, firstly because its an awsome team, and secondly, some of the best moments of my college life revolve around iGEM.<br><br />
My research interests include Developmental Biology, Immunology and Neurobiology. I have worked on 2 summer projects, one on "Developing DNA microarray test for identifying HIV-1 subtypes" and the other one was on " Role of Hesr-1 and Hesr2 genes in Mouse Myocardium". My other hobbies include cooking, making friends, DANCING, reading novels etc. Besides my acads, I am exploring spirituality, though its just a start now.</p><br><br />
Email: I am available at <a href="mailto:harsh.balu@gmail.com">harsh.balu@gmail.com</a><br><br />
</td><br />
<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/f/fe/Harsh1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Pawan Kumar</font></b><br />
<p CLASS="justifyalign">Hello Everyone. I am presently in 4th year of my Dual Degree Program at Dept. of Biotechnology,IIT Madras. I am interested in Computational Biology, as mathematics is what I like. My recently concluded work is "Analysis of navigation of honey bee on the basis of <a href="http://en.wikipedia.org/wiki/Levy_flight"><font color="0000FF">Levy flight</font></a>, under the guidance of Dr. Dr. h.c.Randolf Menzel, Freie University, Berlin. While working for iGEM I realized Mol Bio is not my cup of tea.<br>I also have keen interest in Economics, History and Political Science. I am hard core fan of Cricket and this is the only sports which I play. I love cooking, traveling, writing poems (ob! Hindi),old Hindi melodies. </p><br />
Email:<a href="mailto:pawan.singhiitm@gmail.com">pawan.singhiitm@gmail.com</a><br><br />
</td><br />
<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/6/62/Pawan1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Rama Krishna</font></b><br />
<p CLASS="justifyalign"><br />
Hello, hmmmmmmmm.<br><br />
Currently in fourth year, I am, pursuing a B.Tech in Biotechnology at IIT Madras. Herh herh herh.<br><br />
Being an undergraduate, all the time in the world to pursue my hobbies like a profession, I have, which include synthetic biology, photography (shooting the skies, I like and making time-lapse movies, I love), cycling, trekking and trying to understand evolution and its implications. <br><br />
Being an undergraduate, I am poor enough to not have a lab of my own to make cells do some really crazy stuff I would like them do, to not have an Olympus E3 with some really awesome f/1.4 super tele-lens, to not have a Trek 5900 and to not have some pro camping gear.<br><br />
Having not wasted any summers of my undergraduate life, I have managed to build a cool vertical take-off and landing vehicle, simulate how HIV-1 protease moves around in a solution (Bangalore is a lovely place to be in during summers) and program cells to behave like a clock with a customized period (oh! I also happened to be New York that summer and I just can't live without spicy Indian food. The <a href="https://digamma.cs.unm.edu/wiki/bin/view/McogPublicWeb/WebHome"><font color="0000FF">lab</font></a> i worked in made a tic-tac-toe playing machine with RNA molecules! how cool is that?). I am pretty much hooked up with making cells do some crazy stuff, mostly fancy, which people would like to use it elsewhere. <br />
</p><br />
Email:<a href="ramakrishna.89@gmail.com">ramakrishna.89@gmail.com</a><br><br />
Click <a href="http://openwetware.org/wiki/User:Rama_Krishna"><font color="0000FF">here</font></a> for my webpage.<br />
</td><br />
<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/5/54/Rama1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Srivats V</font></b><br />
<p CLASS="justifyalign">Greetings, synthetic biologists and Stumblers alike. I am currently in my fourth year, pursuing a dual degree in Biotechnology at IIT Madras. I am primarily a molecular biologist and have previously worked on The mechanisms of Puf5p mediated translational repression in <i>S. cerevisiae</i> and a sequence/structure analysis of Puf5 binding to the HO mRNA 3’ UTR in Dr. Wickens' lab at the University of Wisconsin, Madison(Summer 2009).<br><br><br />
That apart, I am an avid quizzer, fan of Queen, Star Wars, and His Noodliness, the Flying Spaghetti Monster. My ambition in life is to one day have an Erdős–Bacon number of under 6, and I am one of those who sincerely believe that the answer to Life, the Universe and everything is 42! See you at the Jamboree. Bring a towel.</p><br><br />
Email:<a href="mailto:srivats88@gmail.com">srivats88@gmail.com</a><br><br />
</td><br />
<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/8/8e/Srivats1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Swathi Ayloo</font></b><br />
<p CLASS="justifyalign">Hi! I am a final year undergrad student interested in Molecular and Cell Biology. My summer project this year at the Max-Planck Institute for Molecular Cell Biology and Genetics in Tony and Joe's labs made Biophysics and Systems Biology my new found interests which I am all set to pursue through graduate studies. It has been a great experience working as a team for iGEM 2009. Though I took some time in the beginning to get the hang of synthetic biology and how networks work, it was a complete learning experience and more importantly lot of fun.<br><br />
If I am not busy with science, you'll find me playing around with Python (something which I began recently), listening to Beatles, reading books, look around to grab some coffee or just having fun. All set to meet you guys at the Jamboree!</p><br><br />
Email: <a href="mailto:swathiayloo@gmail.com">swathiayloo@gmail.com</a><br><br />
</td><br />
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<img src="https://static.igem.org/mediawiki/2009/3/3e/Swathi1.jpg" align="right" width="150" height="150"></a><br />
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{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/Team_underTeam:IIT Madras/Team under2009-10-22T00:20:42Z<p>Ramakrishna: </p>
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<b><font color="#000">Abdul Majeed</font></b><br />
<p CLASS="justifyalign">I am currently in my fourth year, pursuing a dual degree in Bioengineering at IIT Madras. I did my schooling at Dubai and then came to India for professional studies. Well, my research interests include Systems Biology and Biochemical engineering. But since the past 8 months while working for iGEM 2009, I developed a passion for Synthetic Biology. My past research experience include working for Dr.Reed's group on Constrain based network analysis of sugar fermentation in <i>Saccharomyces Cerevisiae</i> at University of Wisconsin, Madison(Summer 2009) and an Industrial experience at Dr. Reddys Laboratory(Summer 2008). I enjoy playing and watching soccer. I aim high in life and try to keep up to my set standards. See you guys at the Jamboree!</p><br />
Email:<a href="mailto:m.abdul.majeed1@gmail.com">m.abdul.majeed1@gmail.com</a><br><br />
Click <a href="http://openwetware.org/wiki/User:Mohammed_Abdul_Majeed"><font color="0000FF">here</font></a> for my webpage.<br />
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<b><font color="#000">Ajit Kamath</font></b><br />
<p CLASS="justifyalign">I am from Mumbai, India. I love playing with cells and making them do our bidding. Apart from synthetic biology, I am also interested in Developmental Immunology. I was in Bonn, Germany for the last summer studying Eosinophil activation and development in mice.</p> <br />
<p CLASS="justifyalign">I shall finish my Masters from IIT Madras in 2011. Apart from playing with cells, I like playing with computers. Some of my hobbies include cooking, reading, writing blogs and music. </p><br />
<p CLASS="justifyalign">My Biggest and Dearest Hobby: Cracking the vaguest of Jokes. Ask my Team mates for confirmation. ;)</p><br />
<p CLASS="justifyalign">To hear some of them buzz me at <a href="mailto:ajit9988@gmail.com">ajit9988@gmail.com</a></p><br />
<br><br />
</td><br />
<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/1/19/Ajit1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Arun Murali</font></b><br />
<p CLASS="justifyalign">Hello World. Currently in my fourth year in the Department of Biotechnology, I am primarily interested in Molecular Biology. As for my research experience, I was in Germany this summer working on certain cell death pathways and interaction studies between proteins involved in the pathways.<br><br />
<br />
Coming to my hobbies and other eccentricities, I play Football and Table Tennis, participate in a range of Literary activities (quizzes, Creative writing, Dramatics, word games etc), and also listen to most kinds of music, though I prefer alternative metal. I also have a vague fascination with the number 19. Oh and the Flying Spaghetti Monster…. His Noodliness Rocks!<br><br />
<br />
P.S. Chuck Norris has ATP, only for him it is Adenosine Tetra Phosphate! (One of those lab nights where we were waiting for a gel to run, right Srivats?)</p><br />
Email:<a href="mailto:arun19murali@gmail.com">arun19murali@gmail.com</a><br><br />
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<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/5/55/Arun1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Bhavya B</font></b><br />
<p CLASS="justifyalign">Hi! Iam currently in fourth year, pursuing a Dual Degree from the Dept of Biotechnology, IIT Madras.I had a great time working for iGEM 2009. It helped me in gaining knowledge about synthetic biology,which adds the spirit of engineering to biology.Working in labs was never more fun and I am gonna miss all the lab hours and the night outs!<br><br />
As for my research experience, I spent this summer(May-July 2009) working as an intern in BIOCON (India) in the cell culture lab where i studied the amino acid consumption patterns of mammalian (CHO) cells.<br><br />
Academics aside, i love music, dancing, spending lazy days with food and fictions, trying my hand at cooking and most of all shopping!!Looking forward to see you all at the Jamboree! </p><br />
Email:<a href="mailto:brundavanambhavya@gmail.com">brundavanambhavya@gmail.com</a><br><br />
</td><br />
<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/2/2c/Bhavya1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Harshvardhan</font></b><br />
<p CLASS="justifyalign">Namaste everyone,<br />
This is Harsh a.k.a takla, yogi, bokka etc. I dont like boasting about myself, because i am so intelligent and smart that I don't need to talk about it ;) Just kidding, but seriously ;) ;) <br><br />
I feel lucky to be in the iGEM'09 team of IITMadras, firstly because its an awsome team, and secondly, some of the best moments of my college life revolve around iGEM.<br><br />
My research interests include Developmental Biology, Immunology and Neurobiology. I have worked on 2 summer projects, one on "Developing DNA microarray test for identifying HIV-1 subtypes" and the other one was on " Role of Hesr-1 and Hesr2 genes in Mouse Myocardium". My other hobbies include cooking, making friends, DANCING, reading novels etc. Besides my acads, I am exploring spirituality, though its just a start now.</p><br><br />
Email: I am available at <a href="mailto:harsh.balu@gmail.com">harsh.balu@gmail.com</a><br><br />
</td><br />
<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/f/fe/Harsh1.jpg" align="right" width="150" height="150"></a><br />
</td><br />
</tr><br />
<tr height="20"></tr><br />
<br />
<tr><br />
<td width="630"><br />
<b><font color="#000">Pawan Kumar</font></b><br />
<p CLASS="justifyalign">Hello Everyone. I am presently in 4th year of my Dual Degree Program at Dept. of Biotechnology,IIT Madras. I am interested in Computational Biology, as mathematics is what I like. My recently concluded work is "Analysis of navigation of honey bee on the basis of <a href="http://en.wikipedia.org/wiki/Levy_flight"><font color="0000FF">Levy flight</font></a>, under the guidance of Dr. Dr. h.c.Randolf Menzel, Freie University, Berlin. While working for iGEM I realized Mol Bio is not my cup of tea.<br>I also have keen interest in Economics, History and Political Science. I am hard core fan of Cricket and this is the only sports which I play. I love cooking, traveling, writing poems (ob! Hindi),old Hindi melodies. </p><br />
Email:<a href="mailto:pawan.singhiitm@gmail.com">pawan.singhiitm@gmail.com</a><br><br />
</td><br />
<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/6/62/Pawan1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Rama Krishna</font></b><br />
<p CLASS="justifyalign"><br />
Hello, hmmmmmmmm.<br><br />
Currently in fourth year, I am, pursuing a B.Tech in Biotechnology at IIT Madras. Herh herh herh.<br><br />
Being an undergraduate, all the time in the world to pursue my hobbies like a profession, I have, which include synthetic biology, photography (shooting the skies, I like and making time-lapse movies, I love), cycling, trekking and trying to understand evolution and its implications. <br><br />
Being an undergraduate, I am poor enough to not have a lab of my own to make cells do some really crazy stuff I would like them do, to not have an Olympus E3 with some really awesome f/1.4 super tele-lens, to not have a Trek 5900 and to not have some pro camping gear.<br><br />
Having not wasted any summers of my undergraduate life, I have managed to build a cool vertical take-off and landing vehicle, simulate how HIV-1 protease moves around in a solution (Bangalore is a lovely place to be in during summers) and program cells to behave like a clock with a customized period (oh! I also happened to be New York that summer and I just can't live without spicy Indian food. The <a href="http://www.cumc.columbia.edu/dept/medicine/BasicResearchers/Stojanovic.html"><font color="0000FF">lab</font></a> i worked in made a tic-tac-toe playing machine with RNA molecules! how cool is that?). I am pretty much hooked up with making cells do some crazy stuff, mostly fancy, which people would like to use it elsewhere. <br />
</p><br />
Email:<a href="ramakrishna.89@gmail.com">ramakrishna.89@gmail.com</a><br><br />
Click <a href="http://openwetware.org/wiki/User:Rama_Krishna"><font color="0000FF">here</font></a> for my webpage.<br />
</td><br />
<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/5/54/Rama1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Srivats V</font></b><br />
<p CLASS="justifyalign">Greetings, synthetic biologists and Stumblers alike. I am currently in my fourth year, pursuing a dual degree in Biotechnology at IIT Madras. I am primarily a molecular biologist and have previously worked on The mechanisms of Puf5p mediated translational repression in <i>S. cerevisiae</i> and a sequence/structure analysis of Puf5 binding to the HO mRNA 3’ UTR in Dr. Wickens' lab at the University of Wisconsin, Madison(Summer 2009).<br><br><br />
That apart, I am an avid quizzer, fan of Queen, Star Wars, and His Noodliness, the Flying Spaghetti Monster. My ambition in life is to one day have an Erdős–Bacon number of under 6, and I am one of those who sincerely believe that the answer to Life, the Universe and everything is 42! See you at the Jamboree. Bring a towel.</p><br><br />
Email:<a href="mailto:srivats88@gmail.com">srivats88@gmail.com</a><br><br />
</td><br />
<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/8/8e/Srivats1.jpg" align="right" width="150" height="150"></a><br />
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<b><font color="#000">Swathi Ayloo</font></b><br />
<p CLASS="justifyalign">Hi! I am a final year undergrad student interested in Molecular and Cell Biology. My summer project this year at the Max-Planck Institute for Molecular Cell Biology and Genetics in Tony and Joe's labs made Biophysics and Systems Biology my new found interests which I am all set to pursue through graduate studies. It has been a great experience working as a team for iGEM 2009. Though I took some time in the beginning to get the hang of synthetic biology and how networks work, it was a complete learning experience and more importantly lot of fun.<br><br />
If I am not busy with science, you'll find me playing around with Python (something which I began recently), listening to Beatles, reading books, look around to grab some coffee or just having fun. All set to meet you guys at the Jamboree!</p><br><br />
Email: <a href="mailto:swathiayloo@gmail.com">swathiayloo@gmail.com</a><br><br />
</td><br />
<td width="170"><br />
<img src="https://static.igem.org/mediawiki/2009/3/3e/Swathi1.jpg" align="right" width="150" height="150"></a><br />
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{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ProjectTeam:IIT Madras/Project2009-10-21T14:42:57Z<p>Ramakrishna: /* Expected behavior of the system */</p>
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<p CLASS="justifyalign">Our project PLASMID - Plasmid Locking Assembly for Sustaining Multiple Inserted DNA - introduces a new paradigm in gene regulation. The study is based on the concept of plasmid loss. Any episome (extrachromosomal genetic element) introduced into the cell shows a segregational asymmetry accompanied with differential growth rates in the absence and presence of episome leading to an overall loss of the episomal unit in the absence of any maintaining selective pressure. It is hypothesized that by appropriately controlling the external selective pressures, one can control the direction of plasmid loss in the cell, modifying the existing gene regulation system in a pre determined manner. It is also hypothesized that introducing negative selective pressures against certain other directions of plasmid loss, in the form of constitutively repressed endotoxins will help streamline the regulatory system even further. <br />
<br />
<p CLASS="justifyalign">If successful, this study allows for exquisitely delicate and precise multifactorial regulation of gene control in the future. This model can also be used to hide genes of commercial interest to protect it from unauthorized use (under some conditions).</p><br />
<br />
<br />
<br />
</body><br />
</html><br />
<br />
<br />
==Summary==<br />
<br />
<p CLASS="justifyalign">Our project is based on the fundamental concept of plasmid instability in a novel way to conceal information or ‘lock’ a gene’s function in a cell until the correct combination of inputs is fed into the cell. We call this a ‘combinatorial lock’ or PLASMID. It involves the positive regulation of the gene of interest only on receiving the correct inputs from the user. We use plasmids which can confer resistance to certain antibiotics in the medium and link them up in a certain way (i.e, essentially designing a genetic circuit) so that they repress the expression of the gene of our interest. As the selection pressure is lifted from the media, the plasmids which have the repressors for the gene of interest are lost, hence revealing the gene on using the correct series of antibiotic washes. In essence, the process of unlocking would simply be the correct sequence of antibiotic media in which the cells should be washed.</p><br />
<br />
<p CLASS="justifyalign">We would be working with a 2 plasmid system and it is easy to see that this principle, theoretically, could be extended to N plasmids. In general the code length required to "unlock" is N-1 if the number of plasmids introduced are N. In our case, since the number of plasmids being introduced are 2, the code would essentially be just 1 unit long. Particularly in this case, the 1 unit of code corresponds to growing the cells in one correct antibiotic medium.<br />
</p><br />
<br />
<html><br></html><br />
<br />
[[Image:plasmid network.jpg|650px|center]]<br />
<br />
<html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 1:</font></b><i>The gene of interest is "locked" or repressed by the inhibitor in the plasmid 2. The plasmids are linked up in a certain way that the plasmids need to be lost only in a very specific order, else the cells die due to the release of a toxin. Thus the "unlocking" of the gene of interest requires a predetermined order of growth conditions which allows for a directional loss of the plasmids, and hence the repressor for the promoter that expresses the gene of interest.</i><br />
</p><br />
<br />
<html><br></html><html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
However, in the experiments we have not incorporated any particular gene to be repressed. Instead we study how can we achieve a directed loss of plasmids which is the idea central to the working of the system. In place of a gene of interest, we have placed fluorescent reporters in each plasmid to monitor the presence or absence of any particular plasmid.</p><br />
<br />
==Theory==<br />
<br />
<p CLASS="justifyalign">The most important idea behind the working of the lock is plasmid loss due to lack of selection. Any extra-chromosomal genetic material introduced into the cell tends to disappear over the generations, unless it confers a selective survival advantage over the cells that do not possess the plasmid. During the growth of bacteria, plasmid-free variants arise in the initially homogeneous plasmid-bearing cell population basically in two ways. First, each plasmid-bearing cell has a certain probability to give rise to a plasmid-free cell at cell division (this depends on the mechanisms of plasmid distribution between daughter cells, plasmid copy number at the cell division, the presence of multimer resolution loci, etc.).</p><br />
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[[Image:segregationandplasmidloss.jpg|300px|center]] <br />
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<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 2.1:</font></b> <i>During cell division, rarely all the plasmids segregate into only one of the daughter cells, thus giving rise to plasmid free cells in the population. This event happens independent of what kind of plasmid the cell contains.</i></p><br />
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[[Image:growthcurves.jpg|300px|center]]<br />
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<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 2.2:</font></b><i>Cells containing plasmids have a higher metabolic strain of synthesizing the proteins that are encoded by the plasmid, thus they tend to grow slower than the cells without any plasmids. The growth difference is pronounced especially during the exponential phase.</i></p><br />
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<br />
<p CLASS="justifyalign">Usually, such probability is very low for natural plasmids (about 10-7) whereas recombinant plasmids (i.e. genetically modified) may segregate with a higher probability (10-3—10-5). Several hypotheses have been put forward to explain this tremendous difference: impaired copy number control, the absence/impairment of the multimer resolution genes and random distribution of plasmid among daughter cells for low copy number plasmids. <br />
Second, it was experimentally found that plasmid-bearing cells usually have a lower maximum specific growth rate than their plasmid-free counterparts, and once a plasmid-free cell arises, it competes with its plasmid-bearing counterparts rapidly and ultimately phases it out. Since most recombinant plasmids are not conjugative (not capable of self-transfer to other plasmid-free cells), if a cell has lost a plasmid, there is no way for the cell to acquire it again. Thus, a segregation of plasmids at cell division and the difference in the growth rates of plasmid-free and plasmid bearing subpopulations determine the rate at which plasmids are lost during prolonged cultivation. Here, we use plasmids which can confer resistance to certain antibiotics in the medium and link them up in a certain way so that they repress the expression of the gene of our interest. As the selection pressure is removed, the plasmids which have the repressors for the gene of interest are lost, hence revealing the gene on using the correct series of antibiotic washes.</p><br />
<br />
[[Image:expressionofgeneofinterest.jpg|500px|center]]<br />
<br />
<html><br></html><br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 2.3:</font></b><i>In the initial state, all the cells bear a network of plasmids which can sustain each other under the influence of a particular selection pressure. In this state, the plasmids other than the one containing the gene of interest have repressors for the promoter that expresses the gene of interest in plasmid 1. As the cells are grown in a correct order of varying selection pressures, the cells lose plasmids directionally, thus leading to the expression or "unlocking" of the gene of interest.</i></p><br />
<br />
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<br />
==Circuit==<br />
<br />
<p CLASS="justifyalign">One of the plasmids will have the gene of interest which will be expressed only when the plasmids apart from this particular one are lost in a particular order. This particular order or “code” will consist of a sequence of antibiotic treatments given to the transformed cells. The correct code triggers the loss of plasmids in the cells in a particular order. Each plasmid will be linked to the plasmids which are supposed to be lost before and after it in a highly regulated fashion. The plasmid loss can be regulated very tightly using a “suicide gene” (gene coding for the bacterial gyrase poison). These genes will be triggered when the culture is subjected to the wrong antibiotic (Out of sequence). To ensure that the suicide genes don’t fire randomly, they are under the control of repressors which are on the plasmids that are supposed to be lost after it and are expressed constitutively. Thus, they fire only when the plasmid containing the repressor is lost. When all the plasmids other than the plasmid containing the gene of interest are lost, the repressors that have been blocking the required gene are lost, thus allowing its expression, which can manifest as a phenotype or function in the cell. This will be the “unlocking” of the lock.</p><br />
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----<br />
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The grand idea of a 3 plasmid locking system revolved around constructing something like the below:<br />
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[[Image:3-plasmid system.jpg|650px|center]] <br />
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<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 3.1:</font></b> <i>This is a general circuit for a 3 plasmid system which can show locking property with a code length of 2 units - Ab2->Ab1. This means that the cells need to be grown first in antibiotic medium 2 and then transferred to a medium containing antibiotic 1. It is easy to see that to maintain the whole system of 3 plasmids, the cells need to be grown in medium containing antibiotic 3.</i></p><br />
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To which we thought we could fit in the following parts (the gene of interest is not included. It could be anything):<br />
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[[Image:constructs3plasmids.jpg|700px|center]]<br />
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<b><font color="#000">Fig 3.2:</font></b><i>The 3 plasmid system.</i><br />
<br />
----<br />
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Then we scaled it down and began to build constructs for a 2 plasmid system which works like this:<br />
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[[Image:2-plasmid system.jpg|650px|center]]<br />
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<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 3.3:</font></b><i>This is a general circuit for a 2 plasmid system that can show locking property with a code length of 1 unit - which means the system is unlocked when it is grown in a medium containing antibiotic 1.</i></p><br />
<br />
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To which we fit in parts from the registry to make it look like this:<br />
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[[Image:constructs2plasmids.jpg|600px|center]]<br />
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<b><font color="#000">Fig 3.4:</font></b><i>The 2 plasmid system.</i></p><br />
<br />
----<br />
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For the proof of concept experiments, we built the following constructs:<br />
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[[Image:k272001.jpg|center]]<br />
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[http://partsregistry.org/Part:BBa_K272001 K272001-constitutive RFP expressor]<br />
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<b><font color="#000">Fig 3.5:</font></b><i>This plasmid confers constitutive expression of RFP in the transformed cells.</i><br />
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[[Image:k272002.jpg|center]]<br />
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[http://partsregistry.org/Part:BBa_K272002 K272002-constitutive CFP expressor]<br />
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<b><font color="#000">Fig 3.5:</font></b><i>This plasmid confers constitutive expression of CFP in the transformed cells.</i><br />
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==Expected behavior of the system==<br />
<br />
<br />
== Working with the constructs made for the 2 plasmid lock case ==<br />
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<b><font color="#000">Fig 4.1: The cultures are grown in a medium containing Ampicillin to direct the loss of the plasmid 2 so that the gene of interest is expressed as the repressor for the gene of interest lies in the plasmid 2. </font></b>[[Image:2plasmidcase-1.jpg|650px|center]]<br />
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<br />
<b><font color="#000">Fig 4.2: The cultures are grown first in medium containing Chloramphenicol and then transferred to a medium containing Ampicillin. This means that the medium containing Chloramphenicol is requried to maintain both the plasmdis while growth in Ampicillin is required for unlocking the gene of interest </font></b>[[Image:2plasmidcase-2.jpg|650px|center]]<br />
<br />
== Working with the constructs for the proof of concept ==<br />
<br />
==Work Plan==<br />
<br />
===Building the constructs===<br />
<br />
We have been following 3 different strategies to build our constructs.<br />
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1. The 3A assembly<br />
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[[Image:3Aassembly.jpg|400px|center]]<br />
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<br />
2. The 2A or Standard Assembly<br />
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[[Image:standardassembly.jpg|350px|center]]<br />
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<br />
3. PCR based approach: We plan to proceed with the experiment using a PCR based amplification system, followed by a specific restriction digest and subsequent ligation.<br />
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<br />
This strategy saves a lot of time in cloning the parts together. All it takes is a PCR, a purification step, digestion and ligation. This strategy takes much less time in comparision with the traditional cloning steps using bacteria. Moreover, this process can be automated to ligate a huge number of parts together in a short period of time. It saves the labor of dealing with bacteria for amplifying the required insert after each ligation. <br />
<html><br></html><br />
[[Image:PCR strategy.jpg|750px|center]]<br />
<br />
===Working with the construct===<br />
<br />
<p CLASS="justifyalign">We will be performing a proof-of-concept experiment using the 2 plasmids - K272001 and K272002. The cells will be co-transformed with the plasmids. Each of these plasmids have a different fluorescent reporter and their backbones have different antibiotic resistances. However, the origins of replication in both the plasmids are the same, thus making the plasmids compete with each other in order to remain in the cell based on the selection pressures. The bacteria "titrates" the number of plasmids with a certain origin of replication before each replication. Thus if there are a couple of plasmids with the same origin of replication, and one of them happens to be non essential and does not confer any advantage to the cell for a given growth condition it will eventually be phased out from the population. This is essentially mimicking natural selection. The fluorescent reporters will help in tracing which plasmid is phasing out for a given set of growth conditions.</p><br />
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<br />
<p CLASS="justifyalign">The cotransformed cells will be grown in different antibiotic media (in our case - Ampicillin, Chloramphenicol and both Ampicillin and Chloramphenicol) and the loss of each plasmid will be studied using fluorescent markers under a microscope. Presently we are working with the K272001 (constitutive RFP) in pSB1C3 and K272002 (constitutive CFP) in pSB1A2. We expect to see that the co-transformed cells when grown in a medium with both chloramphenicol and ampicillin should have more number of cells with both the fluorescent reporters than with those which are grown in medium containing either or none of the antibiotics. Specifically, if the cells are grown in medium containing only ampicillin, then the number of cells without CFP should increase and when grown in the absence of chloramphenicol, the number of cells without RFP should be on the rise.</p><br />
<br />
[[Image:proof-of-concept.jpg|650px|center]]<br />
<br />
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<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 6:</font></b><i>The cotransformed cells when grown in different antibiotic media, show different fates. For instance, when the cells are grown in medium containing Ampicillin but not Chloramphenicol, the lack of selection pressure due to chloramphenicol on the cells leads to a gradual phasing out of those cells which contain both the plasmids. Only those with the Ampicillin selection marker eventually dominate the population.</i></p><br />
<br />
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<br />
The constructs required to demonstrate the locking property are being built and will be tested once the construction is done.<br />
<br />
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<br />
==Experiments==<br />
<br />
===Growth curves===<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
<br />
==Results==<br />
<br />
===Growth curves===<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ProjectTeam:IIT Madras/Project2009-10-21T14:39:43Z<p>Ramakrishna: /* Expected behavior of the system */</p>
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<div>{{:Team:IITM/main}}<br />
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<p CLASS="justifyalign">Our project PLASMID - Plasmid Locking Assembly for Sustaining Multiple Inserted DNA - introduces a new paradigm in gene regulation. The study is based on the concept of plasmid loss. Any episome (extrachromosomal genetic element) introduced into the cell shows a segregational asymmetry accompanied with differential growth rates in the absence and presence of episome leading to an overall loss of the episomal unit in the absence of any maintaining selective pressure. It is hypothesized that by appropriately controlling the external selective pressures, one can control the direction of plasmid loss in the cell, modifying the existing gene regulation system in a pre determined manner. It is also hypothesized that introducing negative selective pressures against certain other directions of plasmid loss, in the form of constitutively repressed endotoxins will help streamline the regulatory system even further. <br />
<br />
<p CLASS="justifyalign">If successful, this study allows for exquisitely delicate and precise multifactorial regulation of gene control in the future. This model can also be used to hide genes of commercial interest to protect it from unauthorized use (under some conditions).</p><br />
<br />
<br />
<br />
</body><br />
</html><br />
<br />
<br />
==Summary==<br />
<br />
<p CLASS="justifyalign">Our project is based on the fundamental concept of plasmid instability in a novel way to conceal information or ‘lock’ a gene’s function in a cell until the correct combination of inputs is fed into the cell. We call this a ‘combinatorial lock’ or PLASMID. It involves the positive regulation of the gene of interest only on receiving the correct inputs from the user. We use plasmids which can confer resistance to certain antibiotics in the medium and link them up in a certain way (i.e, essentially designing a genetic circuit) so that they repress the expression of the gene of our interest. As the selection pressure is lifted from the media, the plasmids which have the repressors for the gene of interest are lost, hence revealing the gene on using the correct series of antibiotic washes. In essence, the process of unlocking would simply be the correct sequence of antibiotic media in which the cells should be washed.</p><br />
<br />
<p CLASS="justifyalign">We would be working with a 2 plasmid system and it is easy to see that this principle, theoretically, could be extended to N plasmids. In general the code length required to "unlock" is N-1 if the number of plasmids introduced are N. In our case, since the number of plasmids being introduced are 2, the code would essentially be just 1 unit long. Particularly in this case, the 1 unit of code corresponds to growing the cells in one correct antibiotic medium.<br />
</p><br />
<br />
<html><br></html><br />
<br />
[[Image:plasmid network.jpg|650px|center]]<br />
<br />
<html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 1:</font></b><i>The gene of interest is "locked" or repressed by the inhibitor in the plasmid 2. The plasmids are linked up in a certain way that the plasmids need to be lost only in a very specific order, else the cells die due to the release of a toxin. Thus the "unlocking" of the gene of interest requires a predetermined order of growth conditions which allows for a directional loss of the plasmids, and hence the repressor for the promoter that expresses the gene of interest.</i><br />
</p><br />
<br />
<html><br></html><html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
However, in the experiments we have not incorporated any particular gene to be repressed. Instead we study how can we achieve a directed loss of plasmids which is the idea central to the working of the system. In place of a gene of interest, we have placed fluorescent reporters in each plasmid to monitor the presence or absence of any particular plasmid.</p><br />
<br />
==Theory==<br />
<br />
<p CLASS="justifyalign">The most important idea behind the working of the lock is plasmid loss due to lack of selection. Any extra-chromosomal genetic material introduced into the cell tends to disappear over the generations, unless it confers a selective survival advantage over the cells that do not possess the plasmid. During the growth of bacteria, plasmid-free variants arise in the initially homogeneous plasmid-bearing cell population basically in two ways. First, each plasmid-bearing cell has a certain probability to give rise to a plasmid-free cell at cell division (this depends on the mechanisms of plasmid distribution between daughter cells, plasmid copy number at the cell division, the presence of multimer resolution loci, etc.).</p><br />
<br />
<html><br></html><br />
<html><br></html><br />
<br />
[[Image:segregationandplasmidloss.jpg|300px|center]] <br />
<br />
<html><br></html> <br />
<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 2.1:</font></b> <i>During cell division, rarely all the plasmids segregate into only one of the daughter cells, thus giving rise to plasmid free cells in the population. This event happens independent of what kind of plasmid the cell contains.</i></p><br />
<br />
<html><br></html><br />
<html><br></html><br />
<br />
[[Image:growthcurves.jpg|300px|center]]<br />
<br />
<html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 2.2:</font></b><i>Cells containing plasmids have a higher metabolic strain of synthesizing the proteins that are encoded by the plasmid, thus they tend to grow slower than the cells without any plasmids. The growth difference is pronounced especially during the exponential phase.</i></p><br />
<br />
<html><br></html><br />
<html><br></html><br />
<br />
<p CLASS="justifyalign">Usually, such probability is very low for natural plasmids (about 10-7) whereas recombinant plasmids (i.e. genetically modified) may segregate with a higher probability (10-3—10-5). Several hypotheses have been put forward to explain this tremendous difference: impaired copy number control, the absence/impairment of the multimer resolution genes and random distribution of plasmid among daughter cells for low copy number plasmids. <br />
Second, it was experimentally found that plasmid-bearing cells usually have a lower maximum specific growth rate than their plasmid-free counterparts, and once a plasmid-free cell arises, it competes with its plasmid-bearing counterparts rapidly and ultimately phases it out. Since most recombinant plasmids are not conjugative (not capable of self-transfer to other plasmid-free cells), if a cell has lost a plasmid, there is no way for the cell to acquire it again. Thus, a segregation of plasmids at cell division and the difference in the growth rates of plasmid-free and plasmid bearing subpopulations determine the rate at which plasmids are lost during prolonged cultivation. Here, we use plasmids which can confer resistance to certain antibiotics in the medium and link them up in a certain way so that they repress the expression of the gene of our interest. As the selection pressure is removed, the plasmids which have the repressors for the gene of interest are lost, hence revealing the gene on using the correct series of antibiotic washes.</p><br />
<br />
[[Image:expressionofgeneofinterest.jpg|500px|center]]<br />
<br />
<html><br></html><br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 2.3:</font></b><i>In the initial state, all the cells bear a network of plasmids which can sustain each other under the influence of a particular selection pressure. In this state, the plasmids other than the one containing the gene of interest have repressors for the promoter that expresses the gene of interest in plasmid 1. As the cells are grown in a correct order of varying selection pressures, the cells lose plasmids directionally, thus leading to the expression or "unlocking" of the gene of interest.</i></p><br />
<br />
<html><br></html><br />
<br />
==Circuit==<br />
<br />
<p CLASS="justifyalign">One of the plasmids will have the gene of interest which will be expressed only when the plasmids apart from this particular one are lost in a particular order. This particular order or “code” will consist of a sequence of antibiotic treatments given to the transformed cells. The correct code triggers the loss of plasmids in the cells in a particular order. Each plasmid will be linked to the plasmids which are supposed to be lost before and after it in a highly regulated fashion. The plasmid loss can be regulated very tightly using a “suicide gene” (gene coding for the bacterial gyrase poison). These genes will be triggered when the culture is subjected to the wrong antibiotic (Out of sequence). To ensure that the suicide genes don’t fire randomly, they are under the control of repressors which are on the plasmids that are supposed to be lost after it and are expressed constitutively. Thus, they fire only when the plasmid containing the repressor is lost. When all the plasmids other than the plasmid containing the gene of interest are lost, the repressors that have been blocking the required gene are lost, thus allowing its expression, which can manifest as a phenotype or function in the cell. This will be the “unlocking” of the lock.</p><br />
<br />
<br />
----<br />
<html><br></html><br />
<br />
The grand idea of a 3 plasmid locking system revolved around constructing something like the below:<br />
<br />
<html><br></html><br />
<html><br></html><br />
<br />
[[Image:3-plasmid system.jpg|650px|center]] <br />
<br />
<html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 3.1:</font></b> <i>This is a general circuit for a 3 plasmid system which can show locking property with a code length of 2 units - Ab2->Ab1. This means that the cells need to be grown first in antibiotic medium 2 and then transferred to a medium containing antibiotic 1. It is easy to see that to maintain the whole system of 3 plasmids, the cells need to be grown in medium containing antibiotic 3.</i></p><br />
<br />
<html><br></html><br />
<html><br></html><br />
<br />
To which we thought we could fit in the following parts (the gene of interest is not included. It could be anything):<br />
<br />
<html><br></html><br />
<br />
[[Image:constructs3plasmids.jpg|700px|center]]<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Fig 3.2:</font></b><i>The 3 plasmid system.</i><br />
<br />
----<br />
<html><br></html><br />
Then we scaled it down and began to build constructs for a 2 plasmid system which works like this:<br />
<br />
<html><br></html><br />
<br />
[[Image:2-plasmid system.jpg|650px|center]]<br />
<br />
<html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 3.3:</font></b><i>This is a general circuit for a 2 plasmid system that can show locking property with a code length of 1 unit - which means the system is unlocked when it is grown in a medium containing antibiotic 1.</i></p><br />
<br />
<html><br></html><br />
<html><br></html><br />
<br />
To which we fit in parts from the registry to make it look like this:<br />
<br />
<html><br></html><br />
<br />
[[Image:constructs2plasmids.jpg|600px|center]]<br />
<br />
<html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 3.4:</font></b><i>The 2 plasmid system.</i></p><br />
<br />
----<br />
<br />
<html><br></html><br />
<br />
For the proof of concept experiments, we built the following constructs:<br />
<br />
<html><br></html><br />
<br />
[[Image:k272001.jpg|center]]<br />
<html><br></html><br />
[http://partsregistry.org/Part:BBa_K272001 K272001-constitutive RFP expressor]<br />
<html><br></html><br />
<html><br></html><br />
<br />
<b><font color="#000">Fig 3.5:</font></b><i>This plasmid confers constitutive expression of RFP in the transformed cells.</i><br />
<br />
<html><br></html><br />
<br />
[[Image:k272002.jpg|center]]<br />
<html><br></html><br />
[http://partsregistry.org/Part:BBa_K272002 K272002-constitutive CFP expressor]<br />
<html><br></html><br />
<html><br></html><br />
<br />
<b><font color="#000">Fig 3.5:</font></b><i>This plasmid confers constitutive expression of CFP in the transformed cells.</i><br />
<br />
<html><br></html><br />
<html><br></html><br />
<html><br></html><br />
<br />
==Expected behavior of the system==<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Fig 4.1: The cultures are grown in a medium containing Ampicillin to direct the loss of the plasmid 2 so that the gene of interest is expressed as the repressor for the gene of interest lies in the plasmid 2. </font></b>[[Image:2plasmidcase-1.jpg|650px|center]]<br />
<br />
<html><br></html><br />
<html><br></html><br />
<br />
<b><font color="#000">Fig 4.2: The cultures are grown first in medium containing Chloramphenicol and then transferred to a medium containing Ampicillin. This means that the medium containing Chloramphenicol is requried to maintain both the plasmdis while growth in Ampicillin is required for unlocking the gene of interest </font></b>[[Image:2plasmidcase-2.jpg|650px|center]]<br />
<br />
==Work Plan==<br />
<br />
===Building the constructs===<br />
<br />
We have been following 3 different strategies to build our constructs.<br />
<html><br></html><br />
<br />
1. The 3A assembly<br />
<html><br></html><br />
[[Image:3Aassembly.jpg|400px|center]]<br />
<html><br></html><br />
<br />
2. The 2A or Standard Assembly<br />
<html><br></html><br />
[[Image:standardassembly.jpg|350px|center]]<br />
<html><br></html><br />
<br />
3. PCR based approach: We plan to proceed with the experiment using a PCR based amplification system, followed by a specific restriction digest and subsequent ligation.<br />
<html><br></html><br />
<br />
This strategy saves a lot of time in cloning the parts together. All it takes is a PCR, a purification step, digestion and ligation. This strategy takes much less time in comparision with the traditional cloning steps using bacteria. Moreover, this process can be automated to ligate a huge number of parts together in a short period of time. It saves the labor of dealing with bacteria for amplifying the required insert after each ligation. <br />
<html><br></html><br />
[[Image:PCR strategy.jpg|750px|center]]<br />
<br />
===Working with the construct===<br />
<br />
<p CLASS="justifyalign">We will be performing a proof-of-concept experiment using the 2 plasmids - K272001 and K272002. The cells will be co-transformed with the plasmids. Each of these plasmids have a different fluorescent reporter and their backbones have different antibiotic resistances. However, the origins of replication in both the plasmids are the same, thus making the plasmids compete with each other in order to remain in the cell based on the selection pressures. The bacteria "titrates" the number of plasmids with a certain origin of replication before each replication. Thus if there are a couple of plasmids with the same origin of replication, and one of them happens to be non essential and does not confer any advantage to the cell for a given growth condition it will eventually be phased out from the population. This is essentially mimicking natural selection. The fluorescent reporters will help in tracing which plasmid is phasing out for a given set of growth conditions.</p><br />
<html><br></html><br />
<br />
<p CLASS="justifyalign">The cotransformed cells will be grown in different antibiotic media (in our case - Ampicillin, Chloramphenicol and both Ampicillin and Chloramphenicol) and the loss of each plasmid will be studied using fluorescent markers under a microscope. Presently we are working with the K272001 (constitutive RFP) in pSB1C3 and K272002 (constitutive CFP) in pSB1A2. We expect to see that the co-transformed cells when grown in a medium with both chloramphenicol and ampicillin should have more number of cells with both the fluorescent reporters than with those which are grown in medium containing either or none of the antibiotics. Specifically, if the cells are grown in medium containing only ampicillin, then the number of cells without CFP should increase and when grown in the absence of chloramphenicol, the number of cells without RFP should be on the rise.</p><br />
<br />
[[Image:proof-of-concept.jpg|650px|center]]<br />
<br />
<html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 6:</font></b><i>The cotransformed cells when grown in different antibiotic media, show different fates. For instance, when the cells are grown in medium containing Ampicillin but not Chloramphenicol, the lack of selection pressure due to chloramphenicol on the cells leads to a gradual phasing out of those cells which contain both the plasmids. Only those with the Ampicillin selection marker eventually dominate the population.</i></p><br />
<br />
<html><br></html><br />
<br />
The constructs required to demonstrate the locking property are being built and will be tested once the construction is done.<br />
<br />
<html><br></html><br />
<br />
==Experiments==<br />
<br />
===Growth curves===<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
<br />
==Results==<br />
<br />
===Growth curves===<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ProjectTeam:IIT Madras/Project2009-10-21T14:36:20Z<p>Ramakrishna: /* Expected behavior of the system */</p>
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<div>{{:Team:IITM/main}}<br />
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<p CLASS="justifyalign">Our project PLASMID - Plasmid Locking Assembly for Sustaining Multiple Inserted DNA - introduces a new paradigm in gene regulation. The study is based on the concept of plasmid loss. Any episome (extrachromosomal genetic element) introduced into the cell shows a segregational asymmetry accompanied with differential growth rates in the absence and presence of episome leading to an overall loss of the episomal unit in the absence of any maintaining selective pressure. It is hypothesized that by appropriately controlling the external selective pressures, one can control the direction of plasmid loss in the cell, modifying the existing gene regulation system in a pre determined manner. It is also hypothesized that introducing negative selective pressures against certain other directions of plasmid loss, in the form of constitutively repressed endotoxins will help streamline the regulatory system even further. <br />
<br />
<p CLASS="justifyalign">If successful, this study allows for exquisitely delicate and precise multifactorial regulation of gene control in the future. This model can also be used to hide genes of commercial interest to protect it from unauthorized use (under some conditions).</p><br />
<br />
<br />
<br />
</body><br />
</html><br />
<br />
<br />
==Summary==<br />
<br />
<p CLASS="justifyalign">Our project is based on the fundamental concept of plasmid instability in a novel way to conceal information or ‘lock’ a gene’s function in a cell until the correct combination of inputs is fed into the cell. We call this a ‘combinatorial lock’ or PLASMID. It involves the positive regulation of the gene of interest only on receiving the correct inputs from the user. We use plasmids which can confer resistance to certain antibiotics in the medium and link them up in a certain way (i.e, essentially designing a genetic circuit) so that they repress the expression of the gene of our interest. As the selection pressure is lifted from the media, the plasmids which have the repressors for the gene of interest are lost, hence revealing the gene on using the correct series of antibiotic washes. In essence, the process of unlocking would simply be the correct sequence of antibiotic media in which the cells should be washed.</p><br />
<br />
<p CLASS="justifyalign">We would be working with a 2 plasmid system and it is easy to see that this principle, theoretically, could be extended to N plasmids. In general the code length required to "unlock" is N-1 if the number of plasmids introduced are N. In our case, since the number of plasmids being introduced are 2, the code would essentially be just 1 unit long. Particularly in this case, the 1 unit of code corresponds to growing the cells in one correct antibiotic medium.<br />
</p><br />
<br />
<html><br></html><br />
<br />
[[Image:plasmid network.jpg|650px|center]]<br />
<br />
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<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 1:</font></b><i>The gene of interest is "locked" or repressed by the inhibitor in the plasmid 2. The plasmids are linked up in a certain way that the plasmids need to be lost only in a very specific order, else the cells die due to the release of a toxin. Thus the "unlocking" of the gene of interest requires a predetermined order of growth conditions which allows for a directional loss of the plasmids, and hence the repressor for the promoter that expresses the gene of interest.</i><br />
</p><br />
<br />
<html><br></html><html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
However, in the experiments we have not incorporated any particular gene to be repressed. Instead we study how can we achieve a directed loss of plasmids which is the idea central to the working of the system. In place of a gene of interest, we have placed fluorescent reporters in each plasmid to monitor the presence or absence of any particular plasmid.</p><br />
<br />
==Theory==<br />
<br />
<p CLASS="justifyalign">The most important idea behind the working of the lock is plasmid loss due to lack of selection. Any extra-chromosomal genetic material introduced into the cell tends to disappear over the generations, unless it confers a selective survival advantage over the cells that do not possess the plasmid. During the growth of bacteria, plasmid-free variants arise in the initially homogeneous plasmid-bearing cell population basically in two ways. First, each plasmid-bearing cell has a certain probability to give rise to a plasmid-free cell at cell division (this depends on the mechanisms of plasmid distribution between daughter cells, plasmid copy number at the cell division, the presence of multimer resolution loci, etc.).</p><br />
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[[Image:segregationandplasmidloss.jpg|300px|center]] <br />
<br />
<html><br></html> <br />
<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 2.1:</font></b> <i>During cell division, rarely all the plasmids segregate into only one of the daughter cells, thus giving rise to plasmid free cells in the population. This event happens independent of what kind of plasmid the cell contains.</i></p><br />
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[[Image:growthcurves.jpg|300px|center]]<br />
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<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 2.2:</font></b><i>Cells containing plasmids have a higher metabolic strain of synthesizing the proteins that are encoded by the plasmid, thus they tend to grow slower than the cells without any plasmids. The growth difference is pronounced especially during the exponential phase.</i></p><br />
<br />
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<html><br></html><br />
<br />
<p CLASS="justifyalign">Usually, such probability is very low for natural plasmids (about 10-7) whereas recombinant plasmids (i.e. genetically modified) may segregate with a higher probability (10-3—10-5). Several hypotheses have been put forward to explain this tremendous difference: impaired copy number control, the absence/impairment of the multimer resolution genes and random distribution of plasmid among daughter cells for low copy number plasmids. <br />
Second, it was experimentally found that plasmid-bearing cells usually have a lower maximum specific growth rate than their plasmid-free counterparts, and once a plasmid-free cell arises, it competes with its plasmid-bearing counterparts rapidly and ultimately phases it out. Since most recombinant plasmids are not conjugative (not capable of self-transfer to other plasmid-free cells), if a cell has lost a plasmid, there is no way for the cell to acquire it again. Thus, a segregation of plasmids at cell division and the difference in the growth rates of plasmid-free and plasmid bearing subpopulations determine the rate at which plasmids are lost during prolonged cultivation. Here, we use plasmids which can confer resistance to certain antibiotics in the medium and link them up in a certain way so that they repress the expression of the gene of our interest. As the selection pressure is removed, the plasmids which have the repressors for the gene of interest are lost, hence revealing the gene on using the correct series of antibiotic washes.</p><br />
<br />
[[Image:expressionofgeneofinterest.jpg|500px|center]]<br />
<br />
<html><br></html><br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 2.3:</font></b><i>In the initial state, all the cells bear a network of plasmids which can sustain each other under the influence of a particular selection pressure. In this state, the plasmids other than the one containing the gene of interest have repressors for the promoter that expresses the gene of interest in plasmid 1. As the cells are grown in a correct order of varying selection pressures, the cells lose plasmids directionally, thus leading to the expression or "unlocking" of the gene of interest.</i></p><br />
<br />
<html><br></html><br />
<br />
==Circuit==<br />
<br />
<p CLASS="justifyalign">One of the plasmids will have the gene of interest which will be expressed only when the plasmids apart from this particular one are lost in a particular order. This particular order or “code” will consist of a sequence of antibiotic treatments given to the transformed cells. The correct code triggers the loss of plasmids in the cells in a particular order. Each plasmid will be linked to the plasmids which are supposed to be lost before and after it in a highly regulated fashion. The plasmid loss can be regulated very tightly using a “suicide gene” (gene coding for the bacterial gyrase poison). These genes will be triggered when the culture is subjected to the wrong antibiotic (Out of sequence). To ensure that the suicide genes don’t fire randomly, they are under the control of repressors which are on the plasmids that are supposed to be lost after it and are expressed constitutively. Thus, they fire only when the plasmid containing the repressor is lost. When all the plasmids other than the plasmid containing the gene of interest are lost, the repressors that have been blocking the required gene are lost, thus allowing its expression, which can manifest as a phenotype or function in the cell. This will be the “unlocking” of the lock.</p><br />
<br />
<br />
----<br />
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<br />
The grand idea of a 3 plasmid locking system revolved around constructing something like the below:<br />
<br />
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[[Image:3-plasmid system.jpg|650px|center]] <br />
<br />
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<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 3.1:</font></b> <i>This is a general circuit for a 3 plasmid system which can show locking property with a code length of 2 units - Ab2->Ab1. This means that the cells need to be grown first in antibiotic medium 2 and then transferred to a medium containing antibiotic 1. It is easy to see that to maintain the whole system of 3 plasmids, the cells need to be grown in medium containing antibiotic 3.</i></p><br />
<br />
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To which we thought we could fit in the following parts (the gene of interest is not included. It could be anything):<br />
<br />
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<br />
[[Image:constructs3plasmids.jpg|700px|center]]<br />
<br />
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<br />
<b><font color="#000">Fig 3.2:</font></b><i>The 3 plasmid system.</i><br />
<br />
----<br />
<html><br></html><br />
Then we scaled it down and began to build constructs for a 2 plasmid system which works like this:<br />
<br />
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<br />
[[Image:2-plasmid system.jpg|650px|center]]<br />
<br />
<html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 3.3:</font></b><i>This is a general circuit for a 2 plasmid system that can show locking property with a code length of 1 unit - which means the system is unlocked when it is grown in a medium containing antibiotic 1.</i></p><br />
<br />
<html><br></html><br />
<html><br></html><br />
<br />
To which we fit in parts from the registry to make it look like this:<br />
<br />
<html><br></html><br />
<br />
[[Image:constructs2plasmids.jpg|600px|center]]<br />
<br />
<html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 3.4:</font></b><i>The 2 plasmid system.</i></p><br />
<br />
----<br />
<br />
<html><br></html><br />
<br />
For the proof of concept experiments, we built the following constructs:<br />
<br />
<html><br></html><br />
<br />
[[Image:k272001.jpg|center]]<br />
<html><br></html><br />
[http://partsregistry.org/Part:BBa_K272001 K272001-constitutive RFP expressor]<br />
<html><br></html><br />
<html><br></html><br />
<br />
<b><font color="#000">Fig 3.5:</font></b><i>This plasmid confers constitutive expression of RFP in the transformed cells.</i><br />
<br />
<html><br></html><br />
<br />
[[Image:k272002.jpg|center]]<br />
<html><br></html><br />
[http://partsregistry.org/Part:BBa_K272002 K272002-constitutive CFP expressor]<br />
<html><br></html><br />
<html><br></html><br />
<br />
<b><font color="#000">Fig 3.5:</font></b><i>This plasmid confers constitutive expression of CFP in the transformed cells.</i><br />
<br />
<html><br></html><br />
<html><br></html><br />
<html><br></html><br />
<br />
==Expected behavior of the system==<br />
<br />
<html><br></html><br />
<br />
<b><font color="#000">Fig 4.1: The cultures are grown in a medium containing Ampicillin to direct the loss of the Chloramphenicol backbone plasmid so that the gene of interest is expressed. </font></b>[[Image:2plasmidcase-1.jpg|650px|center]]<br />
<br />
<html><br></html><br />
<html><br></html><br />
<br />
<b><font color="#000">Fig 4.2: The cultures are grown first in medium containing Chloramphenicol and then transferred to a medium containing Ampicillin. This means that the medium containing Chloramphenicol is requried to maintain both the plasmdis while growth in Ampicillin is required for unlocking the gene of interest </font></b>[[Image:2plasmidcase-2.jpg|650px|center]]<br />
<br />
==Work Plan==<br />
<br />
===Building the constructs===<br />
<br />
We have been following 3 different strategies to build our constructs.<br />
<html><br></html><br />
<br />
1. The 3A assembly<br />
<html><br></html><br />
[[Image:3Aassembly.jpg|400px|center]]<br />
<html><br></html><br />
<br />
2. The 2A or Standard Assembly<br />
<html><br></html><br />
[[Image:standardassembly.jpg|350px|center]]<br />
<html><br></html><br />
<br />
3. PCR based approach: We plan to proceed with the experiment using a PCR based amplification system, followed by a specific restriction digest and subsequent ligation.<br />
<html><br></html><br />
<br />
This strategy saves a lot of time in cloning the parts together. All it takes is a PCR, a purification step, digestion and ligation. This strategy takes much less time in comparision with the traditional cloning steps using bacteria. Moreover, this process can be automated to ligate a huge number of parts together in a short period of time. It saves the labor of dealing with bacteria for amplifying the required insert after each ligation. <br />
<html><br></html><br />
[[Image:PCR strategy.jpg|750px|center]]<br />
<br />
===Working with the construct===<br />
<br />
<p CLASS="justifyalign">We will be performing a proof-of-concept experiment using the 2 plasmids - K272001 and K272002. The cells will be co-transformed with the plasmids. Each of these plasmids have a different fluorescent reporter and their backbones have different antibiotic resistances. However, the origins of replication in both the plasmids are the same, thus making the plasmids compete with each other in order to remain in the cell based on the selection pressures. The bacteria "titrates" the number of plasmids with a certain origin of replication before each replication. Thus if there are a couple of plasmids with the same origin of replication, and one of them happens to be non essential and does not confer any advantage to the cell for a given growth condition it will eventually be phased out from the population. This is essentially mimicking natural selection. The fluorescent reporters will help in tracing which plasmid is phasing out for a given set of growth conditions.</p><br />
<html><br></html><br />
<br />
<p CLASS="justifyalign">The cotransformed cells will be grown in different antibiotic media (in our case - Ampicillin, Chloramphenicol and both Ampicillin and Chloramphenicol) and the loss of each plasmid will be studied using fluorescent markers under a microscope. Presently we are working with the K272001 (constitutive RFP) in pSB1C3 and K272002 (constitutive CFP) in pSB1A2. We expect to see that the co-transformed cells when grown in a medium with both chloramphenicol and ampicillin should have more number of cells with both the fluorescent reporters than with those which are grown in medium containing either or none of the antibiotics. Specifically, if the cells are grown in medium containing only ampicillin, then the number of cells without CFP should increase and when grown in the absence of chloramphenicol, the number of cells without RFP should be on the rise.</p><br />
<br />
[[Image:proof-of-concept.jpg|650px|center]]<br />
<br />
<html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
<b><font color="#000">Fig 6:</font></b><i>The cotransformed cells when grown in different antibiotic media, show different fates. For instance, when the cells are grown in medium containing Ampicillin but not Chloramphenicol, the lack of selection pressure due to chloramphenicol on the cells leads to a gradual phasing out of those cells which contain both the plasmids. Only those with the Ampicillin selection marker eventually dominate the population.</i></p><br />
<br />
<html><br></html><br />
<br />
The constructs required to demonstrate the locking property are being built and will be tested once the construction is done.<br />
<br />
<html><br></html><br />
<br />
==Experiments==<br />
<br />
===Growth curves===<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
<br />
==Results==<br />
<br />
===Growth curves===<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ProjectTeam:IIT Madras/Project2009-10-21T14:35:45Z<p>Ramakrishna: /* Expected behavior of the system */</p>
<hr />
<div>{{:Team:IITM/main}}<br />
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<p CLASS="justifyalign">Our project PLASMID - Plasmid Locking Assembly for Sustaining Multiple Inserted DNA - introduces a new paradigm in gene regulation. The study is based on the concept of plasmid loss. Any episome (extrachromosomal genetic element) introduced into the cell shows a segregational asymmetry accompanied with differential growth rates in the absence and presence of episome leading to an overall loss of the episomal unit in the absence of any maintaining selective pressure. It is hypothesized that by appropriately controlling the external selective pressures, one can control the direction of plasmid loss in the cell, modifying the existing gene regulation system in a pre determined manner. It is also hypothesized that introducing negative selective pressures against certain other directions of plasmid loss, in the form of constitutively repressed endotoxins will help streamline the regulatory system even further. <br />
<br />
<p CLASS="justifyalign">If successful, this study allows for exquisitely delicate and precise multifactorial regulation of gene control in the future. This model can also be used to hide genes of commercial interest to protect it from unauthorized use (under some conditions).</p><br />
<br />
<br />
<br />
</body><br />
</html><br />
<br />
<br />
==Summary==<br />
<br />
<p CLASS="justifyalign">Our project is based on the fundamental concept of plasmid instability in a novel way to conceal information or ‘lock’ a gene’s function in a cell until the correct combination of inputs is fed into the cell. We call this a ‘combinatorial lock’ or PLASMID. It involves the positive regulation of the gene of interest only on receiving the correct inputs from the user. We use plasmids which can confer resistance to certain antibiotics in the medium and link them up in a certain way (i.e, essentially designing a genetic circuit) so that they repress the expression of the gene of our interest. As the selection pressure is lifted from the media, the plasmids which have the repressors for the gene of interest are lost, hence revealing the gene on using the correct series of antibiotic washes. In essence, the process of unlocking would simply be the correct sequence of antibiotic media in which the cells should be washed.</p><br />
<br />
<p CLASS="justifyalign">We would be working with a 2 plasmid system and it is easy to see that this principle, theoretically, could be extended to N plasmids. In general the code length required to "unlock" is N-1 if the number of plasmids introduced are N. In our case, since the number of plasmids being introduced are 2, the code would essentially be just 1 unit long. Particularly in this case, the 1 unit of code corresponds to growing the cells in one correct antibiotic medium.<br />
</p><br />
<br />
<html><br></html><br />
<br />
[[Image:plasmid network.jpg|650px|center]]<br />
<br />
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<b><font color="#000">Fig 1:</font></b><i>The gene of interest is "locked" or repressed by the inhibitor in the plasmid 2. The plasmids are linked up in a certain way that the plasmids need to be lost only in a very specific order, else the cells die due to the release of a toxin. Thus the "unlocking" of the gene of interest requires a predetermined order of growth conditions which allows for a directional loss of the plasmids, and hence the repressor for the promoter that expresses the gene of interest.</i><br />
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However, in the experiments we have not incorporated any particular gene to be repressed. Instead we study how can we achieve a directed loss of plasmids which is the idea central to the working of the system. In place of a gene of interest, we have placed fluorescent reporters in each plasmid to monitor the presence or absence of any particular plasmid.</p><br />
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==Theory==<br />
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<p CLASS="justifyalign">The most important idea behind the working of the lock is plasmid loss due to lack of selection. Any extra-chromosomal genetic material introduced into the cell tends to disappear over the generations, unless it confers a selective survival advantage over the cells that do not possess the plasmid. During the growth of bacteria, plasmid-free variants arise in the initially homogeneous plasmid-bearing cell population basically in two ways. First, each plasmid-bearing cell has a certain probability to give rise to a plasmid-free cell at cell division (this depends on the mechanisms of plasmid distribution between daughter cells, plasmid copy number at the cell division, the presence of multimer resolution loci, etc.).</p><br />
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<b><font color="#000">Fig 2.1:</font></b> <i>During cell division, rarely all the plasmids segregate into only one of the daughter cells, thus giving rise to plasmid free cells in the population. This event happens independent of what kind of plasmid the cell contains.</i></p><br />
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<b><font color="#000">Fig 2.2:</font></b><i>Cells containing plasmids have a higher metabolic strain of synthesizing the proteins that are encoded by the plasmid, thus they tend to grow slower than the cells without any plasmids. The growth difference is pronounced especially during the exponential phase.</i></p><br />
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<p CLASS="justifyalign">Usually, such probability is very low for natural plasmids (about 10-7) whereas recombinant plasmids (i.e. genetically modified) may segregate with a higher probability (10-3—10-5). Several hypotheses have been put forward to explain this tremendous difference: impaired copy number control, the absence/impairment of the multimer resolution genes and random distribution of plasmid among daughter cells for low copy number plasmids. <br />
Second, it was experimentally found that plasmid-bearing cells usually have a lower maximum specific growth rate than their plasmid-free counterparts, and once a plasmid-free cell arises, it competes with its plasmid-bearing counterparts rapidly and ultimately phases it out. Since most recombinant plasmids are not conjugative (not capable of self-transfer to other plasmid-free cells), if a cell has lost a plasmid, there is no way for the cell to acquire it again. Thus, a segregation of plasmids at cell division and the difference in the growth rates of plasmid-free and plasmid bearing subpopulations determine the rate at which plasmids are lost during prolonged cultivation. Here, we use plasmids which can confer resistance to certain antibiotics in the medium and link them up in a certain way so that they repress the expression of the gene of our interest. As the selection pressure is removed, the plasmids which have the repressors for the gene of interest are lost, hence revealing the gene on using the correct series of antibiotic washes.</p><br />
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[[Image:expressionofgeneofinterest.jpg|500px|center]]<br />
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<b><font color="#000">Fig 2.3:</font></b><i>In the initial state, all the cells bear a network of plasmids which can sustain each other under the influence of a particular selection pressure. In this state, the plasmids other than the one containing the gene of interest have repressors for the promoter that expresses the gene of interest in plasmid 1. As the cells are grown in a correct order of varying selection pressures, the cells lose plasmids directionally, thus leading to the expression or "unlocking" of the gene of interest.</i></p><br />
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==Circuit==<br />
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<p CLASS="justifyalign">One of the plasmids will have the gene of interest which will be expressed only when the plasmids apart from this particular one are lost in a particular order. This particular order or “code” will consist of a sequence of antibiotic treatments given to the transformed cells. The correct code triggers the loss of plasmids in the cells in a particular order. Each plasmid will be linked to the plasmids which are supposed to be lost before and after it in a highly regulated fashion. The plasmid loss can be regulated very tightly using a “suicide gene” (gene coding for the bacterial gyrase poison). These genes will be triggered when the culture is subjected to the wrong antibiotic (Out of sequence). To ensure that the suicide genes don’t fire randomly, they are under the control of repressors which are on the plasmids that are supposed to be lost after it and are expressed constitutively. Thus, they fire only when the plasmid containing the repressor is lost. When all the plasmids other than the plasmid containing the gene of interest are lost, the repressors that have been blocking the required gene are lost, thus allowing its expression, which can manifest as a phenotype or function in the cell. This will be the “unlocking” of the lock.</p><br />
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The grand idea of a 3 plasmid locking system revolved around constructing something like the below:<br />
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<b><font color="#000">Fig 3.1:</font></b> <i>This is a general circuit for a 3 plasmid system which can show locking property with a code length of 2 units - Ab2->Ab1. This means that the cells need to be grown first in antibiotic medium 2 and then transferred to a medium containing antibiotic 1. It is easy to see that to maintain the whole system of 3 plasmids, the cells need to be grown in medium containing antibiotic 3.</i></p><br />
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To which we thought we could fit in the following parts (the gene of interest is not included. It could be anything):<br />
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<b><font color="#000">Fig 3.2:</font></b><i>The 3 plasmid system.</i><br />
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Then we scaled it down and began to build constructs for a 2 plasmid system which works like this:<br />
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<b><font color="#000">Fig 3.3:</font></b><i>This is a general circuit for a 2 plasmid system that can show locking property with a code length of 1 unit - which means the system is unlocked when it is grown in a medium containing antibiotic 1.</i></p><br />
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To which we fit in parts from the registry to make it look like this:<br />
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<b><font color="#000">Fig 3.4:</font></b><i>The 2 plasmid system.</i></p><br />
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For the proof of concept experiments, we built the following constructs:<br />
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[http://partsregistry.org/Part:BBa_K272001 K272001-constitutive RFP expressor]<br />
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<b><font color="#000">Fig 3.5:</font></b><i>This plasmid confers constitutive expression of RFP in the transformed cells.</i><br />
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[http://partsregistry.org/Part:BBa_K272002 K272002-constitutive CFP expressor]<br />
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<b><font color="#000">Fig 3.5:</font></b><i>This plasmid confers constitutive expression of CFP in the transformed cells.</i><br />
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==Expected behavior of the system==<br />
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<b><font color="#000">Fig 4.1: The cultures are grown in a medium containing Ampicillin to direct the loss of the plasmid 2 so that the gene of interest is expressed. </font></b>[[Image:2plasmidcase-1.jpg|650px|center]]<br />
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<b><font color="#000">Fig 4.2: The cultures are grown first in medium containing Chloramphenicol and then transferred to a medium containing Ampicillin. This means that the medium containing Chloramphenicol is requried to maintain both the plasmdis while growth in Ampicillin is required for unlocking the gene of interest </font></b>[[Image:2plasmidcase-2.jpg|650px|center]]<br />
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==Work Plan==<br />
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===Building the constructs===<br />
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We have been following 3 different strategies to build our constructs.<br />
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1. The 3A assembly<br />
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2. The 2A or Standard Assembly<br />
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3. PCR based approach: We plan to proceed with the experiment using a PCR based amplification system, followed by a specific restriction digest and subsequent ligation.<br />
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This strategy saves a lot of time in cloning the parts together. All it takes is a PCR, a purification step, digestion and ligation. This strategy takes much less time in comparision with the traditional cloning steps using bacteria. Moreover, this process can be automated to ligate a huge number of parts together in a short period of time. It saves the labor of dealing with bacteria for amplifying the required insert after each ligation. <br />
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[[Image:PCR strategy.jpg|750px|center]]<br />
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===Working with the construct===<br />
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<p CLASS="justifyalign">We will be performing a proof-of-concept experiment using the 2 plasmids - K272001 and K272002. The cells will be co-transformed with the plasmids. Each of these plasmids have a different fluorescent reporter and their backbones have different antibiotic resistances. However, the origins of replication in both the plasmids are the same, thus making the plasmids compete with each other in order to remain in the cell based on the selection pressures. The bacteria "titrates" the number of plasmids with a certain origin of replication before each replication. Thus if there are a couple of plasmids with the same origin of replication, and one of them happens to be non essential and does not confer any advantage to the cell for a given growth condition it will eventually be phased out from the population. This is essentially mimicking natural selection. The fluorescent reporters will help in tracing which plasmid is phasing out for a given set of growth conditions.</p><br />
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<p CLASS="justifyalign">The cotransformed cells will be grown in different antibiotic media (in our case - Ampicillin, Chloramphenicol and both Ampicillin and Chloramphenicol) and the loss of each plasmid will be studied using fluorescent markers under a microscope. Presently we are working with the K272001 (constitutive RFP) in pSB1C3 and K272002 (constitutive CFP) in pSB1A2. We expect to see that the co-transformed cells when grown in a medium with both chloramphenicol and ampicillin should have more number of cells with both the fluorescent reporters than with those which are grown in medium containing either or none of the antibiotics. Specifically, if the cells are grown in medium containing only ampicillin, then the number of cells without CFP should increase and when grown in the absence of chloramphenicol, the number of cells without RFP should be on the rise.</p><br />
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[[Image:proof-of-concept.jpg|650px|center]]<br />
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<b><font color="#000">Fig 6:</font></b><i>The cotransformed cells when grown in different antibiotic media, show different fates. For instance, when the cells are grown in medium containing Ampicillin but not Chloramphenicol, the lack of selection pressure due to chloramphenicol on the cells leads to a gradual phasing out of those cells which contain both the plasmids. Only those with the Ampicillin selection marker eventually dominate the population.</i></p><br />
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The constructs required to demonstrate the locking property are being built and will be tested once the construction is done.<br />
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==Experiments==<br />
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===Growth curves===<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
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==Results==<br />
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===Growth curves===<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
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{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T14:26:58Z<p>Ramakrishna: /* Fluorescence Imaging of cells to check for directed plasmid loss */</p>
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==Experiments==<br />
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===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
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<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 7.<br />
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<b><font color="#000">Fig 7: </font></b><i>This experiment helps in comparing the growth rates of various strains in different antibiotic media.</i><br />
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<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
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* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
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<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
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Experimental protocol:<br />
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<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
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<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
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Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
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===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
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<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
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Fig 6.3:Experimental procedure for measuring the rate of plasmid loss by fluorescent imaging<br />
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We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
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The expected behavior of the system is (listed in the same order as the above 3 conditions):<br />
# RFP (pSB1C3)containing Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope and those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# CFP (pSB1A2)containing cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cotransformed cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
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Protocol:<br />
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1. Inoculate 3ml LB containing Ampicillin with a colony of cells transformed with CFP (pSB1A2)- broth 1, inoculate 3ml LB containing Chloramphenicol with a colony of cells transformed with RFP (pSB1C3) - broth 2, inoculate 3ml LB containing Ampicillin and Chloramphenicol with a colony of cells co-transformed with CFP (pSB1A2)and RFP (pSB1C3) - broth 3.<br />
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2. Culture is grown for 4 hours or till the OD600 crosses 0.1.<br />
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3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculate from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculated in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).<br />
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Required inoculations are:<br />
a. Broth 1 into tube with no antibiotic in the LB - tube 1<br />
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b. Broth 1 into tube with Ampicillin in the LB - tube 2<br />
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c. Broth 2 into tube with no antibioti in the LB - tube 3<br />
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d. Broth 2 into tube with Chloramphenicol in the LB - tube 4<br />
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e. Broth 3 into tube no antibiotic in the LB - tube 5<br />
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f. Broth 3 into tube with Ampicillin in the LB - tube 6<br />
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g. Broth 3 into tube with Chloramphenicol in the LB - tube 7<br />
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h. Broth 3 into tube with both the antibiotics in the LB - tube 8<br />
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4. This culture is allowed to grow for 2.5 hours.<br />
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5. Measure the OD at this stage and also prepare a slide for fluorescence imaging from each of the 8 tubes.<br />
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6. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculate from this 2.5 hour culture to each of the fresh 5ml LB medium with the same antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 2.5 hour culture, Vol 1 is the volume of this 2.5 hour culture that needs to be inoculated in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1). i.e. The culture from spent tube 1 is used to inoculte a fresh tube 1. Similarly follow the same for all the other 7 tubes.<br />
7. Let this new culture grow for 2.5 hours and repeat from step 5 for about 5 times.<br />
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Click [https://2009.igem.org/Team:IIT_Madras/Results#Fluorescent_Imaging here] to see the results of the fluorescence imaging. <br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T14:25:33Z<p>Ramakrishna: /* Fluorescence Imaging of cells to check for directed plasmid loss */</p>
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==Experiments==<br />
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===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
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<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 7.<br />
</p><br />
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[[Image:growth curves.jpg|700px]]<br />
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<b><font color="#000">Fig 7: </font></b><i>This experiment helps in comparing the growth rates of various strains in different antibiotic media.</i><br />
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<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
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[[Image:flourescenceimagingexp.jpg|700px]]<br />
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Fig 6.3:<br />
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We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
<br />
<br />
<br />
The expected behavior of the system is (listed in the same order as the above 3 conditions):<br />
# RFP (pSB1C3)containing Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope and those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# CFP (pSB1A2)containing cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cotransformed cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
<br />
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Protocol:<br />
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1. Inoculate 3ml LB containing Ampicillin with a colony of cells transformed with CFP (pSB1A2)- broth 1, inoculate 3ml LB containing Chloramphenicol with a colony of cells transformed with RFP (pSB1C3) - broth 2, inoculate 3ml LB containing Ampicillin and Chloramphenicol with a colony of cells co-transformed with CFP (pSB1A2)and RFP (pSB1C3) - broth 3.<br />
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2. Culture is grown for 4 hours or till the OD600 crosses 0.1.<br />
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3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculate from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculated in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).<br />
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Required inoculations are:<br />
a. Broth 1 into tube with no antibiotic in the LB - tube 1<br />
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b. Broth 1 into tube with Ampicillin in the LB - tube 2<br />
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c. Broth 2 into tube with no antibioti in the LB - tube 3<br />
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d. Broth 2 into tube with Chloramphenicol in the LB - tube 4<br />
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e. Broth 3 into tube no antibiotic in the LB - tube 5<br />
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f. Broth 3 into tube with Ampicillin in the LB - tube 6<br />
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g. Broth 3 into tube with Chloramphenicol in the LB - tube 7<br />
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h. Broth 3 into tube with both the antibiotics in the LB - tube 8<br />
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4. This culture is allowed to grow for 2.5 hours.<br />
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5. Measure the OD at this stage and also prepare a slide for fluorescence imaging from each of the 8 tubes.<br />
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6. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculate from this 2.5 hour culture to each of the fresh 5ml LB medium with the same antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 2.5 hour culture, Vol 1 is the volume of this 2.5 hour culture that needs to be inoculated in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1). i.e. The culture from spent tube 1 is used to inoculte a fresh tube 1. Similarly follow the same for all the other 7 tubes.<br />
7. Let this new culture grow for 2.5 hours and repeat from step 5 for about 5 times.<br />
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<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Fluorescent_Imaging here] to see the results of the fluorescence imaging. <br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/File:Flourescenceimagingexp.jpgFile:Flourescenceimagingexp.jpg2009-10-21T14:25:06Z<p>Ramakrishna: </p>
<hr />
<div></div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ResultsTeam:IIT Madras/Results2009-10-21T14:22:35Z<p>Ramakrishna: /* Comparing the differences in the growth rates of cells with and without plasmids in various media */</p>
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==Results==<br />
<br />
=== Comparing the differences in the growth rates of cells with and without plasmids in various media ===<br />
----<br />
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Growth curves<br />
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DH5a grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
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[[Image:DH5aall4cases.jpg|600px]]<br />
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RFP (pSB1C3) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
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[[Image:rfpall4cases.jpg|600px]]<br />
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CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
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[[Image:cfpall4cases.jpg|600px]]<br />
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RFP (pSB1C3) - CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
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[[Image:RC4cases.jpg|600px]]<br />
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All 4 strains in media without antibiotics<br />
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[[Image:noaball4.jpg|600px]]<br />
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All 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin. <br />
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[[Image:corraball4.jpg|600px]]<br />
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log (OD600) vs Time plot for all 4 strains in media without antibiotics<br />
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[[Image:logall4noab.jpg|600px]]<br />
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log (OD600) vs Time plot for all 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin.<br />
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[[Image:logcorrab.jpg|600px]]<br />
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<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ResultsTeam:IIT Madras/Results2009-10-21T14:21:54Z<p>Ramakrishna: /* Comparing the differences in the growth rates of cells with and without plasmids in various media */</p>
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==Results==<br />
<br />
=== Comparing the differences in the growth rates of cells with and without plasmids in various media ===<br />
----<br />
<br />
Growth curves<br />
<br />
DH5a grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
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[[Image:DH5aall4cases.jpg|600px]]<br />
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RFP (pSB1C3) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
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[[Image:rfpall4cases.jpg|600px]]<br />
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CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
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[[Image:cfpall4cases.jpg|600px]]<br />
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RFP (pSB1C3) - CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
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[[Image:RC4cases.jpg|600px]]<br />
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All 4 strains in media without antibiotics<br />
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[[Image:noaball4.jpg|600px]]<br />
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All 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin. <br />
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[[Image:corraball4.jpg|600px]]<br />
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log (OD600) vs Time plot for all 4 strains in media without antibiotics<br />
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[[Image:logall4noab.jpg]]<br />
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log (OD600) vs Time plot for all 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol and Ampicillin.<br />
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[[Image:logcorrab.jpg]]<br />
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<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ResultsTeam:IIT Madras/Results2009-10-21T14:19:54Z<p>Ramakrishna: /* Comparing the differences in the growth rates of cells with and without plasmids in various media */</p>
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==Results==<br />
<br />
=== Comparing the differences in the growth rates of cells with and without plasmids in various media ===<br />
----<br />
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Growth curves<br />
<br />
DH5a grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
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[[Image:DH5aall4cases.jpg|600px]]<br />
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RFP (pSB1C3) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
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[[Image:rfpall4cases.jpg|600px]]<br />
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CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
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[[Image:cfpall4cases.jpg|600px]]<br />
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RFP (pSB1C3) - CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
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[[Image:RC4cases.jpg|600px]]<br />
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All 4 strains in media without antibiotics<br />
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[[Image:noaball4.jpg|600px]]<br />
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All 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol adn Ampicillin. <br />
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[[Image:corraball4.jpg|600px]]<br />
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<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ResultsTeam:IIT Madras/Results2009-10-21T14:09:56Z<p>Ramakrishna: /* Comparing the differences in the growth rates of cells with and without plasmids in various media */</p>
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==Results==<br />
<br />
=== Comparing the differences in the growth rates of cells with and without plasmids in various media ===<br />
----<br />
<br />
Growth curves<br />
<br />
DH5a grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
[[Image:DH5aall4cases.jpg|600px]]<br />
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RFP (pSB1C3) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
[[Image:rfpall4cases.jpg|600px]]<br />
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CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
[[Image:cfpall4cases.jpg|600px]]<br />
<br />
RFP (pSB1C3) - CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
[[Image:RC4cases.jpg|600px]]<br />
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All 4 strains in media without antibiotics<br />
[[Image:noaball4.jpg|600px]]<br />
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All 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol adn Ampicillin. <br />
[[Image:corraball4.jpg|600px]]<br />
<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ResultsTeam:IIT Madras/Results2009-10-21T14:07:59Z<p>Ramakrishna: /* Comparing the differences in the growth rates of cells with and without plasmids in various media */</p>
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==Results==<br />
<br />
=== Comparing the differences in the growth rates of cells with and without plasmids in various media ===<br />
----<br />
<br />
Growth curves<br />
<br />
DH5a grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
[[Image:DH5aall4cases.jpg]]<br />
<br />
RFP (pSB1C3) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
[[Image:rfpall4cases.jpg]]<br />
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CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
[[Image:cfpall4cases.jpg]]<br />
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RFP (pSB1C3) - CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
[[Image:RC4cases.jpg]]<br />
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All 4 strains in media without antibiotics<br />
[[Image:noaball4.jpg]]<br />
<br />
All 4 strains in their corresponding antibiotic media - DH5a in Lb without antibiotics, RFP (pSB1C3) in LB with Chloramphenicol, CFP (pSB1A2) in LB with Ampicillin, RFP (pSB1C3) - CFP (pSB1A2) cells in LB with Chloramphenicol adn Ampicillin. <br />
[[Image:corraball4.jpg]]<br />
<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/File:DH5aall4cases.jpgFile:DH5aall4cases.jpg2009-10-21T14:04:10Z<p>Ramakrishna: </p>
<hr />
<div></div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ResultsTeam:IIT Madras/Results2009-10-21T14:02:11Z<p>Ramakrishna: /* Comparing the differences in the growth rates of cells with and without plasmids in various media */</p>
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==Results==<br />
<br />
=== Comparing the differences in the growth rates of cells with and without plasmids in various media ===<br />
----<br />
<br />
Growth curves<br />
<br />
DH5a grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
[[Image:DH5aall4cases.jpg]]<br />
<br />
RFP (pSB1C3) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
[[Image:rfpall4cases.jpg]]<br />
<br />
CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
[[Image:cfpall4cases.jpg]]<br />
<br />
RFP (pSB1C3) - CFP (pSB1A2) contanining cells grown in LB containing no antibiotic, LB containing Ampicillin, LB containing Chloramphenicol, LB containing both antibiotics.<br />
[[Image:RC4cases.jpg]]<br />
<br />
===Modeling===<br />
===Fluorescent Imaging===<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T13:57:32Z<p>Ramakrishna: /* Fluorescence Imaging of cells to check for directed plasmid loss */</p>
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 5.<br />
</p><br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<br />
<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
<br />
<br />
<br />
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[[Image:flourescenceimagingexp.jpg]]<br />
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Fig 6.3:<br />
<html><br></html><br />
<br />
<br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
<br />
<br />
<br />
The expected behavior of the system is (listed in the same order as the above 3 conditions):<br />
# RFP (pSB1C3)containing Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope and those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# CFP (pSB1A2)containing cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cotransformed cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
<br />
<br />
<html><br></html><br />
<br />
Protocol:<br />
<html><br></html><br />
1. Inoculate 3ml LB containing Ampicillin with a colony of cells transformed with CFP (pSB1A2)- broth 1, inoculate 3ml LB containing Chloramphenicol with a colony of cells transformed with RFP (pSB1C3) - broth 2, inoculate 3ml LB containing Ampicillin and Chloramphenicol with a colony of cells co-transformed with CFP (pSB1A2)and RFP (pSB1C3) - broth 3.<br />
<html><br></html><br />
2. Culture is grown for 4 hours or till the OD600 crosses 0.1.<br />
<html><br></html><br />
3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculate from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculated in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).<br />
<html><br></html><br />
<html><br></html><br />
Required inoculations are:<br />
a. Broth 1 into tube with no antibiotic in the LB - tube 1<br />
<html><br></html><br />
b. Broth 1 into tube with Ampicillin in the LB - tube 2<br />
<html><br></html><br />
c. Broth 2 into tube with no antibioti in the LB - tube 3<br />
<html><br></html><br />
d. Broth 2 into tube with Chloramphenicol in the LB - tube 4<br />
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e. Broth 3 into tube no antibiotic in the LB - tube 5<br />
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f. Broth 3 into tube with Ampicillin in the LB - tube 6<br />
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g. Broth 3 into tube with Chloramphenicol in the LB - tube 7<br />
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h. Broth 3 into tube with both the antibiotics in the LB - tube 8<br />
<html><br></html><br />
<html><br></html><br />
4. This culture is allowed to grow for 2.5 hours.<br />
<html><br></html><br />
5. Measure the OD at this stage and also prepare a slide for fluorescence imaging from each of the 8 tubes.<br />
<html><br></html><br />
6. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculate from this 2.5 hour culture to each of the fresh 5ml LB medium with the same antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 2.5 hour culture, Vol 1 is the volume of this 2.5 hour culture that needs to be inoculated in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1). i.e. The culture from spent tube 1 is used to inoculte a fresh tube 1. Similarly follow the same for all the other 7 tubes.<br />
7. Let this new culture grow for 2.5 hours and repeat from step 5 for about 5 times.<br />
<html><br></html><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Fluorescent_Imaging here] to see the results of the fluorescence imaging. <br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T13:49:55Z<p>Ramakrishna: /* Fluorescence Imaging of cells to check for directed plasmid loss */</p>
<hr />
<div>{{:Team:IITM/main}}<br />
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 5.<br />
</p><br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<br />
<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
<br />
<br />
<br />
<html><br></html><br />
[[Image:flourescenceimagingexp.jpg]]<br />
<html><br></html><br />
Fig 6.3:<br />
<html><br></html><br />
<br />
<br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
<br />
<br />
<br />
The expected behavior of the system is (listed in the same order as the above 3 conditions):<br />
# RFP (pSB1C3)containing Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope and those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# CFP (pSB1A2)containing cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cotransformed cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
<br />
<br />
<html><br></html><br />
<br />
Protocol:<br />
1. Inoculate 3ml LB containing Ampicillin with a colony of cells transformed with CFP (pSB1A2)- broth 1, inoculate 3ml LB containing Chloramphenicol with a colony of cells transformed with RFP (pSB1C3) - broth 2, inoculate 3ml LB containing Ampicillin and Chloramphenicol with a colony of cells co-transformed with CFP (pSB1A2)and RFP (pSB1C3) - broth 3.<br />
<html><br></html><br />
2. Culture is grown for 4 hours or till the OD600 crosses 0.1.<br />
<html><br></html><br />
3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculate from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculated in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).<br />
<html><br></html><br />
<html><br></html><br />
Required inoculations are:<br />
a. Broth 1 into tube with no antibiotic in the LB - tube 1<br />
<html><br></html><br />
b. Broth 1 into tube with Ampicillin in the LB - tube 2<br />
<html><br></html><br />
c. Broth 2 into tube with no antibioti in the LB - tube 3<br />
<html><br></html><br />
d. Broth 2 into tube with Chloramphenicol in the LB - tube 4<br />
<html><br></html><br />
e. Broth 3 into tube no antibiotic in the LB - tube 5<br />
<html><br></html><br />
f. Broth 3 into tube with Ampicillin in the LB - tube 6<br />
<html><br></html><br />
g. Broth 3 into tube with Chloramphenicol in the LB - tube 7<br />
<html><br></html><br />
h. Broth 3 into tube with both the antibiotics in the LB - tube 8<br />
<html><br></html><br />
<html><br></html><br />
4. This culture is allowed to grow for 2.5 hours.<br />
<html><br></html><br />
5. Measure the OD at this stage and also prepare a slide for fluorescence imaging from each of the 8 tubes.<br />
<html><br></html><br />
6. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculate from this 2.5 hour culture to each of the fresh 5ml LB medium with the same antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 2.5 hour culture, Vol 1 is the volume of this 2.5 hour culture that needs to be inoculated in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1). i.e. The culture from spent tube 1 is used to inoculte a fresh tube 1. Similarly follow the same for all the other 7 tubes.<br />
7. Let this new culture grow for 2.5 hours and repeat from step 5 for about 5 times.<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T13:40:22Z<p>Ramakrishna: /* Experiments */</p>
<hr />
<div>{{:Team:IITM/main}}<br />
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 5.<br />
</p><br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<br />
<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
<br />
<br />
<br />
<html><br></html><br />
[[Image:flourescenceimagingexp.jpg]]<br />
<html><br></html><br />
Fig 6.3:<br />
<html><br></html><br />
<br />
<br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
<br />
<br />
<br />
The expected behavior of the system is (listed in the same order as the above 3 conditions):<br />
# RFP (pSB1C3)containing Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope and those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# CFP (pSB1A2)containing cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cotransformed cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
<br />
<br />
<html><br></html><br />
<br />
Protocol:<br />
# Inoculate 3ml LB containing Ampicillin with a colony of cells transformed with CFP (pSB1A2)- broth 1, inoculate 3ml LB containing Chloramphenicol with a colony of cells transformed with RFP (pSB1C3) - broth 2, inoculate 3ml LB containing Ampicillin and Chloramphenicol with a colony of cells co-transformed with CFP (pSB1A2)and RFP (pSB1C3) - broth 3.<br />
# Culture is grown for 4 hours or till the OD600 crosses 0.1.<br />
# Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculate from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculated in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).<br />
<html><br></html><br />
Required inoculations are:<br />
1. Broth 1 into tube with no antibiotic in the LB<br />
2. Broth 1 into tube with Ampicillin in the LB<br />
3. Broth 2 into tube with no antibioti in the LBc<br />
4. Broth 2 into tube with Chloramphenicol in the LB<br />
5. Broth 3 into tube no antibiotic in the LB<br />
6. Broth 3 into tube with Ampicillin in the LB<br />
7. Broth 3 into tube with Chloramphenicol in the LB<br />
8. Broth 3 into tube with both the antibiotics in the LB<br />
<br />
# This culture is allowed to grow for 2.5 hours.<br />
# Measure the OD at this stage and also prepare a slide for fluorescence imaging from each of the 8 tubes.<br />
# <br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T13:27:41Z<p>Ramakrishna: /* Fluorescence Imaging of cells to check for directed plasmid loss */</p>
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 5.<br />
</p><br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<br />
<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
<br />
<br />
<br />
<html><br></html><br />
[[Image:flourescenceimagingexp.jpg]]<br />
<html><br></html><br />
Fig 6.3:<br />
<html><br></html><br />
<br />
<br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
<br />
<br />
<br />
The expected behavior of the system is (listed in the same order as the above 3 conditions):<br />
# RFP (pSB1C3)containing Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope and those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# CFP (pSB1A2)containing cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cotransformed cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
<br />
<br />
<html><br></html><br />
<br />
Protocol:<br />
# Incoulte 3ml LB containing no antibiotic with a colony of DH5a, inoculate 3ml LB containing Ampicillin with a colony of cells transformed with CFP (pSB1A2), inoculate 3ml LB containing Chloramphenicol with a colony of cells transformed with RFP (pSB1C3), inoculate 3ml LB containing Ampicillin and Chloramphenicol with a colony of cells co-transformed with CFP (pSB1A2)and RFP (pSB1C3).<br />
# Culture is grown for 4 hours or till the OD600 crosses 0.1.<br />
# <br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T13:26:35Z<p>Ramakrishna: /* Experiments */</p>
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<div>{{:Team:IITM/main}}<br />
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 5.<br />
</p><br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<br />
<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<br />
<p CLASS="justifyalign"><br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
</p><br />
<br />
<br />
<html><br></html><br />
[[Image:flourescenceimagingexp.jpg]]<br />
<html><br></html><br />
Fig 6.3:<br />
<html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
</p><br />
<br />
<p CLASS="justifyalign"><br />
The expected behavior of the system is (listed in the same order as the above 3 conditions):<br />
# RFP (pSB1C3)containing Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope and those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# CFP (pSB1A2)containing cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cotransformed cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
</p><br />
<br />
<html><br></html><br />
<br />
<p CLASS="justifyalign"><br />
Protocol:<br />
# Incoulte 3ml LB containing no antibiotic with a colony of DH5a, inoculate 3ml LB containing Ampicillin with a colony of cells transformed with CFP (pSB1A2), inoculate 3ml LB containing Chloramphenicol with a colony of cells transformed with RFP (pSB1C3), inoculate 3ml LB containing Ampicillin and Chloramphenicol with a colony of cells co-transformed with CFP (pSB1A2)and RFP (pSB1C3).<br />
# Culture is grown for 4 hours or till the OD600 crosses 0.1.<br />
# <br />
</p><br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T13:23:49Z<p>Ramakrishna: /* Fluorescence Imaging of cells to check for directed plasmid loss */</p>
<hr />
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 5.<br />
</p><br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<br />
<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
<br />
<html><br></html><br />
[[Image:flourescenceimagingexp.jpg]]<br />
<html><br></html><br />
Fig 6.3:<br />
<html><br></html><br />
<br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
<br />
The expected behavior of the system is (listed in the same order as the above 3 conditions):<br />
# RFP (pSB1C3)containing Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope and those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# CFP (pSB1A2)containing cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cotransformed cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
<br />
<html><br></html><br />
<br />
Protocol:<br />
# Incoulte 3ml LB containing no antibiotic with a colony of DH5a, inoculate 3ml LB containing Ampicillin with a colony of cells transformed with CFP (pSB1A2), inoculate 3ml LB containing Chloramphenicol with a colony of cells transformed with RFP (pSB1C3), inoculate 3ml LB containing Ampicillin and Chloramphenicol with a colony of cells co-transformed with CFP (pSB1A2)and RFP (pSB1C3).<br />
# Let this culture grown for 4 hours or till the OD600 crosses 0.1.<br />
# <br />
<br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T13:20:28Z<p>Ramakrishna: /* Fluorescence Imaging of cells to check for directed plasmid loss */</p>
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 5.<br />
</p><br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<br />
<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
<br />
<html><br></html><br />
[[Image:flourescenceimagingexp.jpg]]<br />
<html><br></html><br />
Fig 6.3:<br />
<html><br></html><br />
<br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
<br />
The expected behavior of the system is (listed in the same order as the above 3 conditions):<br />
# RFP (pSB1C3)containing Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope and those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# CFP (pSB1A2)containing cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cotransformed cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
<br />
<html><br></html><br />
<br />
Protocol:<br />
#<br />
<br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T13:11:39Z<p>Ramakrishna: /* Fluorescence Imaging of cells to check for directed plasmid loss */</p>
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<div>{{:Team:IITM/main}}<br />
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 5.<br />
</p><br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<br />
<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
<br />
<html><br></html><br />
[[Image:flourescenceimagingexp.jpg]]<br />
<html><br></html><br />
Fig 6.3:<br />
<html><br></html><br />
<br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
<br />
The expected behavior of the system is (listed in the same order as the above 3 conditions):<br />
# RFP (pSB1C3)<br />
* Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope <br />
* Cells which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# CFP (pSB1A2)<br />
* Cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence<br />
* Cells which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# <br />
*Cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence<br />
* Cells which are grown in the Ampicillin containing medium would all be fluorescing Cyan<br />
* Cells which are grown in the Chloramphenicol containing medium would all be fluorescing Red<br />
* Cells which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
<br />
<html><br></html><br />
<br />
Protocol:<br />
#<br />
<br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T13:09:25Z<p>Ramakrishna: /* Fluorescence Imaging of cells to check for directed plasmid loss */</p>
<hr />
<div>{{:Team:IITM/main}}<br />
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 5.<br />
</p><br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<br />
<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
<br />
<html><br></html><br />
[[Image:flourescenceimagingexp.jpg]]<br />
<html><br></html><br />
Fig 6.3:<br />
<html><br></html><br />
<br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
<br />
The expected behavior of the system is (listed in the same order as the above 3 conditions):<br />
# *Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope <br />
*those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# Cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
<br />
<html><br></html><br />
<br />
Protocol:<br />
#<br />
<br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T13:03:57Z<p>Ramakrishna: /* Fluorescence Imaging of cells to check for directed plasmid loss */</p>
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 5.<br />
</p><br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<br />
<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
<br />
<html><br></html><br />
[[Image:flourescenceimagingexp.jpg]]<br />
<html><br></html><br />
Fig 6.3:<br />
<html><br></html><br />
<br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
<br />
The expected behavior of the system would be (listed in the same order as the above 3 conditions):<br />
# Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope while those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# Cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
<br />
Protocol:<br />
<br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T13:02:46Z<p>Ramakrishna: /* Experiments */</p>
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
<p CLASS="justifyalign">We wish to compare the growth rate of the cells that are transformed with a plasmid which shows a constitutive expression of a certain protein to that of growth rate of cells which are not transformed with any plasmids. To study this pattern, we need to grow the different strains of cells in various media as shown in the figure 5.<br />
</p><br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<br />
<p CLASS="justifyalign">We inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<html><br></html><br />
[[Image:flourescenceimagingexp.jpg]]<br />
<html><br></html><br />
Fig 6.3:<br />
<html><br></html><br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
<br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
<br />
The expected behavior of the system would be (listed in the same order as the above 3 conditions):<br />
# Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope while those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# Cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
<br />
Protocol:<br />
<br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T12:53:47Z<p>Ramakrishna: /* Fluorescence Imaging of cells to check for directed plasmid loss */</p>
<hr />
<div>{{:Team:IITM/main}}<br />
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<p CLASS="justifyalign">First we inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<html><br></html><br />
[[Image:flourescenceimagingexp.jpg]]<br />
<html><br></html><br />
Fig 6.3:<br />
<html><br></html><br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
<br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
<br />
The expected behavior of the system would be (listed in the same order as the above 3 conditions):<br />
# Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope while those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# Cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
<br />
Protocol:<br />
<br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T12:53:20Z<p>Ramakrishna: /* Fluorescence Imaging of cells to check for directed plasmid loss */</p>
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<p CLASS="justifyalign">First we inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<html><br></html><br />
[[Image:flourescenceimagingexp.jpg]]<br />
Fig 6.3:<br />
<html><br></html><br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
<br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure 6.3. <br />
# RFP (pSB1C3) containing cells will be grown in medium containing no antibiotic and in medium containing Chloramphenicol<br />
# CFP (pSB1A2) containing cells will be grown in medium containing no antibiotic and in medium containing Ampicillin.<br />
# Co-transformed cell will be grown in medium containing no antibiotic, medium containing only Ampicillin, medium containing only Chloramphenicol, medium containing both Ampicillin and Chloramphenicol. <br />
<br />
The expected behavior of the system would be (listed in the same order as the above 3 conditions):<br />
# Cells grown in medium with no Chloramphenicol would eventually lose the plasmid and hence show no Red fluorescence when imaged under the microscope while those which are grown in the chloramphenicol containing medium would all be fluorescing red.<br />
# Cells grown in medium with no Ampicillin would eventually lose the plasmid and hence show no Cyan fluorescence and those which are grown in the Ampicillin containing medium would all be fluorescing Cyan.<br />
# Cells grown in medium with no antibiotic would eventually lose both the plasmids and hence show no red or Cyan fluorescence, those which are grown in the Ampicillin containing medium would all be fluorescing Cyan, those which are grown in the Chloramphenicol containing medium would all be fluorescing Red and those which are grown in medium containing both the Antibiotics would all be fluorescing both Cyan and Red.<br />
<br />
Protocol:<br />
<br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishnahttp://2009.igem.org/Team:IIT_Madras/ExperimentsTeam:IIT Madras/Experiments2009-10-21T12:40:47Z<p>Ramakrishna: /* Fluorescence Imaging of cells to check for directed plasmid loss */</p>
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==Experiments==<br />
<br />
===Comparing the differences in the growth rates of cells with and without plasmids in various media===<br />
<br />
[[Image:growth curves.jpg|700px]]<br />
<br />
Fig 5: ''This experiment helps in comparing the growth rates of various strains in different antibiotic media'' <br />
<br />
<p CLASS="justifyalign">First we inoculate a colony from the plate containing the required strain into its corresponding media (the media with the required antibiotic). That is, DH5a will be inoculated into LB without any antibiotic, RFP (in pSB1C3)containing cells will be inoculated into LB containing Chloramphenicol (Chl), CFP (in pSB1A2)containing cells will be inoculated into LB containing Ampicillin (Amp) and RFP-CFP co-transformed cells into LB containing both the antibiotics. These are then grown for about 4 hours or till they reach an OD600 value between 0.1 to 0.5.</p><br />
<p CLASS="justifyalign">Then the culture of each strain is taken and inoculated into each of the 5ml broths which contains no antibiotic, Amp, Chl and Amp-Chl so that the OD value in all the freshly inoculated tubes is 0.01. Now we have an array of 16 tubes with various possible combinations:<br />
</p><br />
* DH5a in no antibiotic, DH5a in Amp, DH5a in Chl and Dh5a in Amp-Chl<br />
* RFP (pSB1C3) in no antibiotic, RFP (pSB1C3)in Amp, RFP (pSB1C3)in Chl and RFP (pSB1C3)in Amp-Chl<br />
* CFP (pSB1A2) in no antibiotic, CFP (pSB1A2)in Amp, CFP (pSB1A2)in Chl and CFP (pSB1A2)in Amp-Chl<br />
* RFP (1C3)-CFP (1A2) in no antibiotic, RFP (1C3)-CFP (1A2) in Amp, RFP (1C3)-CFP (1A2) in Chl and RFP (1C3)-CFP (1A2)in Amp-Chl<br />
<br />
<p CLASS="justifyalign">Thus the starting point for all the samples is the same - an OD600 of 0.01.<br />
Every hour starting from the point of inoculation, the OD of all the 16 samples is measured. This would give a fair idea of the growth rates of various strains in different antibiotic media.</p><br />
<br />
Experimental protocol:<br />
<br />
<p CLASS="justifyalign">1. A colony of DH5a is inoculated into 3ml LB without any antibiotic in a 50ml centrifuge tube. Similarly, RFP (1C3) colony is inoculated into 3ml LB with Chl, CFP (1A2) colony into 3ml LB with Amp and RFP-CFP colony into 3ml LB with Amp-Chl.</p><br />
<p CLASS="justifyalign">2. This inoculum is allowed to grow for about 4 hours or till the OD600 of each sample crosses 0.1.</p><br />
<p CLASS="justifyalign">3. Then we use OD 1 x Vol 1 = OD 2 x Vol 2 to measure how much to inoculte from this 4 hour culture to each of the fresh 5ml LB medium with different antibiotic combinations so that the starting OD is 0.01. In this case, OD 1 is the OD600 of the 4 hour culture, Vol 1 is the volume of this 4 hour culture that needs to be inoculted in to the fresh 5ml culture, OD 2 is 0.01 (starting OD for all the 5 ml cultures) and the Vol 2 is the final volume (5ml + vol 1).</p><br />
<p CLASS="justifyalign">4. From this freshly inoculated sample, 150ul of the culture is used to measure the OD every hour starting from the point if inoculation. The 150ul of the sample is diluted 5 times to 750ul and then the OD600 is measured.</p><br />
<p CLASS="justifyalign">5. The whole procedure is repeated to check for reproducibility.</p><br />
<br />
<p CLASS="justifyalign">Note: After the initial 4 hour incubation (the 3ml cultures), we used this to inoculate a 5ml culture with the same antibiotic in the medium as in the 3ml culture. The OD600 of this 5ml tube after the inoculation was 0.01. This was then made to grow for 2.5 hours. This is the culture from which we inoculated the final 16 tubes which would then be used for measurements.</p><br />
<br />
Click [https://2009.igem.org/Team:IIT_Madras/Results#Comparing_the_differences_in_the_growth_rates_of_cells_with_and_without_plasmids_in_various_media here] for the results<br />
<br />
===Modeling===<br />
===Fluorescence Imaging of cells to check for directed plasmid loss===<br />
<html><br></html><br />
[[Image:flourescenceimagingexp.jpg]]<br />
'''Fig 6.3 :'''<br />
<html><br></html><br />
<br />
This experiment is designed to study the plasmid loss when cells (transformed with a plasmid) are grown in media without the required selection pressures. Here, we perform 4 different experiments to study how<br />
# Cells transformed with RFP (pSB1C3) lose the plasmid in the absence of Chloramphenicol in the medium<br />
# Cells transformed with CFP (pSB1A2) lose the plasmid in the absence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the RFP (pSB1C3) plasmid when grown in absence of Chloramphenicol and in the presence of Ampicillin in the medium.<br />
# Cell cotransformed with RFP (pSB1C3) and CFP (pSB1A2) lose the CFP (pSB1A2) plasmid when grown in absence of Ampicillin and in the presence of Chloramphenicol in the medium.<br />
<br />
We pick up colonies of each strain and inoculate it first in a medium with the appropriate antibiotic. Then use this to inoculate in specific growth media as show in the figure <br />
<br />
<br />
{{:Team:IITM/footer}}</div>Ramakrishna