Difference between revisions of "Part:BBa K2206006"

Revision as of 14:13, 19 October 2017

Toehold switch for hsa-miR-15b-5p 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-15b-5p (the trigger RNA). The toehold switch is activated by hsa-miR-15b-5p and regulates production of GFP. 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-15b-5p 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-15b-5p.

The transcripts produced by this part are toxic to E. coli. We recommend the use of an inducible non-leaky promoter to allow for amplification in E. coli. For reference, we found Bba_K808000 was too leaky.

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 1938
Illegal SapI site found at 961

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K2206006 short</partinfo>
-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.
+{{Template:CLSB-UK 17 TS|microRNA=hsa-miR-15b-5p|reporter=GFP|outputtype=fluorescence}}
-This part codes for a toehold switch that contains a region that is complementary to the micro-RNA hsa-mir-15b-5p (the trigger RNA). The toehold switch is activated by hsa-mir-15b-5p 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-15b-5p 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-15b-5p.
-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). An inducible promoter is therefore used to allow for amplification of the constructs in E.coli.
-N.B. This part contains a strong RBS sequence.
 <!-- Add more about the biology of this part here