Team:IIT Madras/Parts/Sandbox

From 2010.igem.org

(Difference between revisions)
 
(One intermediate revision not shown)
Line 64: Line 64:
<li><font style="color:green">Kpn1</font>| Constitutive | RBS | GFP | Terminator | <font style="color:green">BamH1</font>
<li><font style="color:green">Kpn1</font>| Constitutive | RBS | GFP | Terminator | <font style="color:green">BamH1</font>
</ul>
</ul>
 +
<br>
 +
<img src = "https://static.igem.org/mediawiki/2010/d/dd/Block_1_-_2.jpg">
 +
<br>
<h5>Block 2</h5>
<h5>Block 2</h5>
<ul>
<ul>
Line 69: Line 72:
<li><font style="color:green">Nhe1</font> | GFP | Terminator | BamH1 (Extension PCR from 1.4
<li><font style="color:green">Nhe1</font> | GFP | Terminator | BamH1 (Extension PCR from 1.4
</ul>
</ul>
 +
<br>
 +
<img src="https://static.igem.org/mediawiki/2010/a/ab/Block_2.jpg">
 +
<br>
 +
<h5>Block 3</h5>
<h5>Block 3</h5>
<ul>
<ul>
Line 76: Line 83:
<li><font style="color:green">Xho1</font> | RFP | Terminator | <font style="color:green">BamH1</font>
<li><font style="color:green">Xho1</font> | RFP | Terminator | <font style="color:green">BamH1</font>
</ul>
</ul>
 +
<br>
 +
<img src="https://static.igem.org/mediawiki/2010/3/30/Block_3.jpg">
 +
<br>
 +
<h5>Final Design</h5>
<h5>Final Design</h5>
<ul>
<ul>
Line 83: Line 94:
<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>
<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>
</ul>
</ul>
 +
<br>
 +
<img src="https://static.igem.org/mediawiki/2010/4/44/IITM_FInal_Design.jpg">
 +
<br>
 +
<h5>Part Submission</h5>
<h5>Part Submission</h5>
<li><font style="color:green">EcoR1 | Not1 | Xba1</font> | NICE | RBS | <font style="color:green">Spe1 | Not1 | Pst1</font>
<li><font style="color:green">EcoR1 | Not1 | Xba1</font> | NICE | RBS | <font style="color:green">Spe1 | Not1 | Pst1</font>
Line 102: Line 117:
<li>Constitutive | SP310mut2 | GFP | Terminator
<li>Constitutive | SP310mut2 | GFP | Terminator
</ul>
</ul>
-
<h5>Block 3''' -'''To test CRE-loxP system</h5>
+
<h5>Block 3 - To test CRE-loxP system</h5>
<ul>
<ul>
<li>Constitutive | RBS | CRE | Terminator | Constitutive | RBS | loxP | GFP | Terminator | loxP | RFP | Terminator
<li>Constitutive | RBS | CRE | Terminator | Constitutive | RBS | loxP | GFP | Terminator | loxP | RFP | Terminator

Latest revision as of 03:22, 28 October 2010

People

This page describes the construction protocol we wished to follow, and also the final design we hoped to achieve.

Legend

  • This color represents the portion to be added by Extension PCR / Inverse PCR / Assembly PCR
  • 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.
  • This represents an apparent restriction site, one that cannot be used due to the sequences' presence in an ORF

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.
Gene of 295 bp to be synthesised by Assembly PCR / LCR.
Block 1
  • Kpn1 | P170 | RBS | HindIII
  • Kpn1 | NICE | RBS | HindIII
  • HindIII| GFP | Terminator | BamH1
  • Kpn1| Constitutive | RBS | GFP | Terminator | BamH1


Block 2
  • Kpn1 | Constitutive | RBS | HindIII | SP310mut2 | Nhe1
  • Nhe1 | GFP | Terminator | BamH1 (Extension PCR from 1.4


Block 3
  • Kpn1 | Constitutive | RBS | HindIII
  • HindIII | CRE-NLS | Terminator | Sac1
  • Sac1 | Constitutive | RBS | loxP | Nhe1 | GFP | Terminator | loxP | Xho1 (by Inverse PCR + Extension PCR
  • Xho1 | RFP | Terminator | BamH1


Final Design
  • Kpn1 | NICE | RBS | HindIII (as made in 1.1)
  • HindIII | CRE-NLS | Terminator | Sac1 (as made in 3.2)
  • Sac1 | loxP | P170 | RBS | SP310mut2 | Xho1 (by Inverse PCR + Extension PCR)
  • Xho1 | SCM | Nhe1 | loxP | Terminator | BamH1


Part Submission
  • EcoR1 | Not1 | Xba1 | NICE | RBS | Spe1 | Not1 | Pst1
  • EcoR1 | Not1 | Xba1 | P170 | RBS | Spe1 | Not1 | Pst1
  • EcoR1 | Not1 | Xba1 | Kpn1 | Constitutive | RBS | HindIII | SP310mut2 | Nhe1 | GFP | Terminator | BamH1 | Spe1 | Not1 | Pst1
  • EcoR1 | Not1 | Xba1 | Kpn1 | Constitutive | RBS | HindIII | CRE | Terminator | Sac1 | Constitutive | RBS | loxP | Sma1 | GFP | Terminator | loxP | Xho1 | RFP | Terminator | BamH1 | Spe1 | Not1 | Xba1
  • EcoR1 | Not1 | Xba1 | Kpn1 | NICE | RBS | HindIII | CRE-NLS | Terminator | Sac1 | loxP | P170 | RBS | SP310mut2 | Xho1 | SCM | BamH1 | loxP | Terminator | Spe1 | Not1 | Pst1

    Proof of Concept

    Block 1 - To characterize promoters against a standard
    • P170 | GFP | Terminator
    • NICE | GFP | Terminator
    • Constitutive | GFP | Terminator
    Block 2 - Test of export efficiency
    • Constitutive | GFP | Terminator
    • Constitutive | SP310mut2 | GFP | Terminator
    Block 3 - To test CRE-loxP system
    • Constitutive | RBS | CRE | Terminator | Constitutive | RBS | loxP | GFP | Terminator | loxP | RFP | Terminator :Acid Tolerance Promoter (P170) | CRE | Terminator | loxP | Consitutive | GFP | Terminator | loxP | RFP | Terminator

    Final Proposed Design

    NICE | CRE | Terminator | loxP | Acid Tolerance Inducer (P170) | Export tag | SCM | loxP | Terminator

    •  

    Retrieved from "http://2010.igem.org/Team:IIT_Madras/Parts/Sandbox"