Team:IIT Madras/Parts/Sandbox

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Latest revision as of 03:22, 28 October 2010

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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

@@ Line 35: / Line 35: @@
 <li> <a href="https://2010.igem.org/Team:IIT_Madras/Parts/Sandbox">Sandbox</a>
 <li> <a href="https://2010.igem.org/Team:IIT_Madras/Parts/Parts_Submitted">Parts Submitted</a>
+</ul>
 </div>
@@ Line 49: / Line 50: @@
 <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>
 <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>
+</ul>
 </small>
 <br>
@@ Line 61: / Line 63: @@
 <li><font style="color:green">HindIII</font>| 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>
+<br>
+<img src = "https://static.igem.org/mediawiki/2010/d/dd/Block_1_-_2.jpg">
+<br>
 <h5>Block 2</h5>
 <ul>
 <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>
 <li><font style="color:green">Nhe1</font> | GFP | Terminator | BamH1 (Extension PCR from 1.4
+</ul>
+<br>
+<img src="https://static.igem.org/mediawiki/2010/a/ab/Block_2.jpg">
+<br>
 <h5>Block 3</h5>
@@ Line 73: / Line 82: @@
 <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
 <li><font style="color:green">Xho1</font> | RFP | Terminator | <font style="color:green">BamH1</font>
+</ul>
+<br>
+<img src="https://static.igem.org/mediawiki/2010/3/30/Block_3.jpg">
+<br>
 <h5>Final Design</h5>
@@ Line 80: / Line 93: @@
 <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)
 <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>
+<br>
+<img src="https://static.igem.org/mediawiki/2010/4/44/IITM_FInal_Design.jpg">
+<br>
 <h5>Part Submission</h5>
@@ Line 94: / Line 111: @@
 <li>NICE | GFP | Terminator
 <li>Constitutive | GFP | Terminator
+</ul>
 <h5>Block 2 - Test of export efficiency</h5>
 <ul>
 <li>Constitutive | GFP | Terminator
 <li>Constitutive | SP310mut2 | GFP | Terminator
+</ul>
-<h5>Block 3''' -'''To test CRE-loxP system</h5>
+<h5>Block 3 - To test CRE-loxP system</h5>
 <ul>
 <li>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
+</ul>
 <h4>Final Proposed Design</h4>