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Team:IIT Madras/Parts/Sandbox
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| - | <li> <a href="2010.igem.org/Team:IIT_Madras/Parts/Sandbox">Sandbox</a> | + | <li> <a href="https://2010.igem.org/Team:IIT_Madras/Parts/Sandbox">Sandbox</a> |
| - | <li> <a href="2010.igem.org/Team:IIT_Madras/Parts/Parts_Submitted">Parts Submitted</a> | + | <li> <a href="https://2010.igem.org/Team:IIT_Madras/Parts/Parts_Submitted">Parts Submitted</a> |
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Revision as of 01:01, 28 October 2010
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
- 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
