Difference between revisions of "Part:BBa K260016"

Revision as of 15:56, 18 October 2009

TT_FRT_GFP. (double terminator, FRT site, optimised GFP)

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 BBa_E0040.

The first part of this FLP recombinase reporter is the P_FRT_dhfr BioBrick (BBa_K260015). They can both be transferred to the pCC2FosM (BBa_K260000) and pCC2FosMB (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 (BBa_K260016) is always the same, whereas the position of P_FRT_dhfr (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 (BBa_K260015), that have specific homology arms for recombineering-mediated transfer of P_FRT_dhfr to defined positions:

BBa_K260004: pMA-@CC2FosMB-00500bp

BBa_K260005: pMA-@CC2FosMB-01000bp

BBa_K260006: pMA-@CC2FosMB-02000bp

BBa_K260007: pMA-@CC2FosMB-05000bp

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 BBa_K260017, which is also a FLP recombinase reporter.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 144
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 144
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 144
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 809

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K260016 short</partinfo>
-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|]].
+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|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.
@@ Line 22: / Line 21: @@
 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.
+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|BBa_K260017]], which is also a FLP recombinase reporter.