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| - | <title>People</title>
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| - | <body width=80% padding-left=4%>
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| - | <div id="secondarylinks">
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| - | <ul>
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| - | <li> <a href="2010.igem.org/Team:IIT_Madras/Project/Sandbox">Sandbox</a>
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| - | <li> <a href="2010.igem.org/Team:IIT_Madras/Project/Parts_Submitted">Parts Submitted</a>
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| - | This page describes the construction protocol we wished to follow, and also the final design we hoped to achieve.
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| - | <div class="clear"></div>
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| - | <small>
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| - | <h4>Legend</h4>
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| - | <ul>
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| - | <li><font style="color:green"> This color represents the portion to be added by Extension PCR / Inverse PCR / Assembly PCR</font>
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| - | <li><font style="color:red"> This color represents sites that lie in the middle of the parts to be submitted. These are placed so that the promoters and the inserts can be switched out.</font>
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| - | <li><font style="color:blue">This represents an apparent restriction site, one that cannot be used due to the sequences' presence in an ORF</font>
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| - | </small>
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| - | <br>
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| - | The following were the primers we designed for conducting our experiments we obtained from other laboratories in our institute. For Monellin, we planned to use Single Chain Monellin obtained from primary literature as the gene-of-interest.
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| - | <br>
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| - | Gene of 295 bp to be synthesised by Assembly PCR / LCR.<br>
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| - | <h5>Block 1</h5>
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| - | <ul>
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| - | <li><font style="color:green">Kpn1</font> | P170 | <font style="color:green">RBS</font> | <font style="color:green">HindIII</font>
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| - | <li><font style="color:green">Kpn1</font> | NICE | <font style="color:green">RBS</font> | <font style="color:green">HindIII</font>
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| - | <li><font style="color:green">HindIII</font>| GFP | Terminator | <font style="color:green">BamH1</font>
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| - | <li><font style="color:green">Kpn1</font>| Constitutive | RBS | GFP | Terminator | <font style="color:green">BamH1</font>
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| - | <h5>Block 2</h5>
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| - | <ul>
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| - | <li><font style="color:green">Kpn1</font> | <font style="color:green">Constitutive</font> | <font style="color:green">RBS</font> | <font style="color:green">HindIII</font> | SP310mut2 | <font style="color:green">Nhe1</font>
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| - | <li><font style="color:green">Nhe1</font> | GFP | Terminator | BamH1 (Extension PCR from 1.4
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| - | <h5>Block 3</h5>
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| - | <ul>
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| - | <li><font style="color:green">Kpn1</font> | Constitutive | RBS | <font style="color:green">HindIII</font>
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| - | <li><font style="color:green">HindIII</font> | CRE-NLS | <font style="color:green">Terminator</font> | <font style="color:green">Sac1</font>
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| - | <li><font style="color:green">Sac1</font> | Constitutive | RBS | <font style="color:green">loxP</font> | <font style="color:green">Nhe1</font> | GFP | Terminator | <font style="color:green">loxP</font> | <font style="color:green">Xho1</font> (by Inverse PCR + Extension PCR
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| - | <li><font style="color:green">Xho1</font> | RFP | Terminator | <font style="color:green">BamH1</font>
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| - | <h5>Final Design</h5>
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| - | <ul>
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| - | <li>Kpn1 | NICE | RBS | HindIII (as made in 1.1)
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| - | <li>HindIII | CRE-NLS | Terminator | Sac1 (as made in 3.2)
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| - | <li><font style="color:green">Sac1</font> | <font style="color:green">loxP</font> | P170 | <font style="color:green">RBS</font> | SP310mut2 | <font style="color:green">Xho1</font> (by Inverse PCR + Extension PCR)
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| - | <li><font style="color:green">Xho1</font> | SCM | <font style="color:green">Nhe1</font> | <font style="color:green">loxP</font> | <font style="color:green">Terminator</font> | <font style="color:green">BamH1</font>
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| - | <h5>Part Submission</h5>
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| - | <li><font style="color:green">EcoR1 | Not1 | Xba1</font> | NICE | RBS | <font style="color:green">Spe1 | Not1 | Pst1</font>
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| - | <li><font style="color:green">EcoR1 | Not1 | Xba1</font> | P170 | RBS | <font style="color:green">Spe1 | Not1 | Pst1</font>
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| - | <li><font style="color:green">EcoR1 | Not1 | Xba1</font> | <font style="color:red">Kpn1</font> | Constitutive | RBS | <font style="color:red">HindIII</font> | SP310mut2 | <font style="color:red">Nhe1</font> | GFP | Terminator | <font style="color:red">BamH1</font> | <font style="color:green">Spe1 | Not1 | Pst1</font>
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| - | <li><font style="color:green">EcoR1 | Not1 | Xba1</font> | <font style="color:red">Kpn1</font> | Constitutive | RBS | <font style="color:red">HindIII</font> | CRE | Terminator | <font style="color:red">Sac1</font> | Constitutive | RBS | loxP | <font style="color:red">Sma1</font> | GFP | Terminator | loxP | <font style="color:red">Xho1</font> | RFP | Terminator | <font style="color:red">BamH1</font> | <font style="color:green">Spe1 | Not1 | Xba1</font>
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| - | <li><font style="color:green">EcoR1</font> | <font style="color:blue">Not1</font> | <font style="color:green">Xba1</font> | <font style="color:red">Kpn1</font> | NICE | RBS | <font style="color:red">HindIII</font> | CRE-NLS | Terminator | <font style="color:red">Sac1</font> | loxP | P170 | RBS | SP310mut2 | <font style="color:red">Xho1</font> | SCM | <font style="color:red">BamH1</font> | loxP | Terminator | <font style="color:green">Spe1</font> | <font style="color:blue">Not1</font> | <font style="color:green">Pst1</font>
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| - | <h4>Proof of Concept</h4>
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| - | <h5>Block 1 - To characterize promoters against a standard</h5>
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| - | <ul>
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| - | <li>P170 | GFP | Terminator
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| - | <li>NICE | GFP | Terminator
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| - | <li>Constitutive | GFP | Terminator
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| - | <h5>Block 2 - Test of export efficiency</h5>
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| - | <ul>
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| - | <li>Constitutive | GFP | Terminator
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| - | <li>Constitutive | SP310mut2 | GFP | Terminator
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| - | <h5>Block 3''' -'''To test CRE-loxP system</h5>
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| - | <ul>
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| - | <li>Constitutive | RBS | CRE | Terminator | Constitutive | RBS | loxP | GFP | Terminator | loxP | RFP | Terminator
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| - | :Acid Tolerance Promoter (P170) | CRE | Terminator | loxP | Consitutive | GFP | Terminator | loxP | RFP | Terminator
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| - | <h4>Final Proposed Design</h4>
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| - | <big><font style="color:red">NICE | CRE | Terminator | loxP | Acid Tolerance Inducer (P170) | Export tag | SCM | loxP | Terminator</font></big>
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| - | </body>
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| - | {{iitm/footbar}}
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