Difference between revisions of "Part:BBa K2206007"

Revision as of 08:50, 3 October 2017

Toehold switch for hsa-miR-27b-3p with GFPmut3b and inducible promoter

Toehold switches are synthetic riboregulators that regulate gene expression post-transcriptionally. Gene expression can be activated in the presence of a cognate single stranded RNA molecule that contains an arbitrary sequence (the trigger RNA). The trigger RNA binds to the switch through base pairing, causing a conformational change that results in translation of the downstream protein coding region.

This part codes for a toehold switch that contains a region that is complementary to the micro-RNA hsa-mir-27b-3p (the trigger RNA). The toehold switch is activated by hsa-mir-27b-3p and regulates production of GFPmut3b. The fluorescence intensity from GFP is proportional to the number of toehold switches activated (as the more switches activated, the greater the amount of GFP is produced), thus indicating the levels of hsa-mir-27b-3p present (as the more micro-RNA there is, the greater the number of switches activated). This part can therefore be used to quantify the levels of hsa-mir-27b-3p.

We believe that the transcripts produced from this part are toxic when expressed at reasonably high concentrations in E.coli (e.g. the concentrations produced from the BBa_J23111 constitutive promoter). We therefore redesigned the constructs to contain an inducible promoter to allow for amplification in E.coli.

N.B. This part contains a strong RBS sequence

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1144
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 979
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1932
Illegal SapI site found at 961

@@ Line 5: / Line 5: @@
 Toehold switches are synthetic riboregulators that regulate gene expression post-transcriptionally. Gene expression can be activated in the presence of a cognate single stranded RNA molecule that contains an arbitrary sequence (the trigger RNA). The trigger RNA binds to the switch through base pairing, causing a conformational change that results in translation of the downstream protein coding region.
-This part codes for a toehold switch that contains a region that is complementary to the micro-RNA hsa-mir-27b-3p (the trigger RNA). The toehold switch is activated by hsa-mir-27b-3p and regulates production of GFPmut3b. Fluorescence intensity of GFP is proportional to the number of toehold switches activated, thus indicating the levels of hsa-mir-27b-3p present. This part can therefore be used to quantify the levels of hsa-mir-27b-3p.
+This part codes for a toehold switch that contains a region that is complementary to the micro-RNA hsa-mir-27b-3p (the trigger RNA). The toehold switch is activated by hsa-mir-27b-3p and regulates production of GFPmut3b. The fluorescence intensity from GFP is proportional to the number of toehold switches activated (as the more switches activated, the greater the amount of GFP is produced), thus indicating the levels of hsa-mir-27b-3p present (as the more micro-RNA there is, the greater the number of switches activated). This part can therefore be used to quantify the levels of hsa-mir-27b-3p.
 We believe that the transcripts produced from this part are toxic when expressed at reasonably high concentrations in E.coli (e.g. the concentrations produced from the BBa_J23111 constitutive promoter). We therefore redesigned the constructs to contain an inducible promoter to allow for amplification in E.coli.