Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Kaj Bernhardt
Date created: 2009-10-17 11:00:00
Date modified: 2015-05-08 01:11:42
TT_FRT_GFP. (double terminator, FRT site, optimised GFP)
| Types | DnaRegion |
| Roles | Intermediate engineered_region |
| Sequences | BBa_K260016_sequence (Version 1) |
Description
This is the second of two parts of a FLP recombinase reporter. It has a double terminator, a translatable FRT site, and a codon-optimised GFP identical in amino acid sequence to [[Part:BBa_E0040|]].The first part of this FLP recombinase reporter is the P_FRT_dhfr BioBrick ([[Part:BBa_K260015|BBa_K260015]]). They can both be transferred to the pCC2FosM ([[Part:BBa_K260000|BBa_K260000]]) and pCC2FosMB ([[Part:BBa_K260001|BBa_K260001]]) backbones, at defined loci to vary the distance between both FRT sites, of which each part has exactly one.
The position of this TT_FRT_GFP BioBrick ([[Part:BBa_K260016|BBa_K260016]]) is always the same, whereas the position of P_FRT_dhfr ([[Part:BBa_K260015|BBa_K260015]]) can be varied to give inter-FRT distances of 500 bp, 1 kb, 2 kb, 5 kb, 10 kb. This is achieved by starting with different backbones to contain the present BioBrick part ([[Part:BBa_K260015|BBa_K260015]]), that have specific homology arms for recombineering-mediated transfer of P_FRT_dhfr to defined positions:
[[Part:BBa_K260004|BBa_K260004]]: pMA-@CC2FosMB-00500bp
[[Part:BBa_K260005|BBa_K260005]]: pMA-@CC2FosMB-01000bp
[[Part:BBa_K260006|BBa_K260006]]: pMA-@CC2FosMB-02000bp
[[Part:BBa_K260007|BBa_K260007]]: pMA-@CC2FosMB-05000bp
[[Part:BBa_K260008|BBa_K260008]]: pMA-RQ-@CC2FosMB-10000bp
This is how the full reporter works: P_FRT_dhfr_(intervening sequence)_TT_FRT-GFP
The genes for GFP (mut3b) and TmpR (dhfr) have no Start-codon and GFP is silenced by the upstream terminator, but TmpR is expressed. If FLP recombinase levels are high enough, recombination between both FRT sites will delete the intervening dhfr gene plus terminator. Now GFP replaces dhfr and comes under the control of the constitutive promoter that was driving expression of an FRT-TmpR fusion protein before. The result is an FRT-GFP fusion protein that turns the cell green.
This reporter is essentially a FLP activity measurement device, where the transfer curve of [FLP]->GFP can be varied by length of the intervening sequence and thus the distance between both FRT sites. It is similar to P_F3_ZeoR_F3_RFP [[Part:BBa_K260017|]], which is also a FLP recombinase reporter.
Notes
A strong promoter was necessary and we use [[Part:BBa_J23100|BBa_J23100]]. An appropriate reading frame for the FRT site had to be found to prevent premature Stop-codons and hydrophobic amino acid residues. Further, the gene encoding GFP (mut3b) was codon optimised for expression in both E. coli and H. sapiens. For potential use in H. sapiens, the CDS were also made free of polyA sites, exon junctions and CpG dinucleotides where possible.Source
This part was synthesised by "Mr Gene" (Geneart) and is available in the original pMA-BsdR-@CC2FosMB plasmid backbone ([[Part:BBa_K260010|]]), but also pCC2FosMB [[Part:BBa_K260001|]], which is already half-way towards constructing the full P_FRT_dhfr_TT_FRT_GFP reporter.| Sequence Annotation | Location | Component / Role(s) |
| BBa_B0015 (double terminator) translated FRT site optimised GFP (mutb3) BBa_K260016 | 1,129 131,164 166,882 1,882 | feature/stem_loop stem_loop sequence_feature feature/misc CDS feature/cds engineered_region feature/BioBrick |