Team:IIT Madras/Project

From 2010.igem.org

• 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 | GFP | Terminator | BamH1 (by Inverse PCR) // Can't design a Primer this big. :# 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 design === {|$ NICE | CRE | Terminator | loxP | Acid Tolerance Inducer (P170) | Export tag | SCM | loxP | Terminator |}

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