"

From 2010.igem.org

== Project Details== === Part Design === ==== PCR Products ==== {| :'''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 :'''SCM''' ::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 |} === The Experiments === === Part 3 === == Results == {{iitm/footbar}}