Difference between revisions of "Part:BBa K2206006"

Latest revision as of 20:47, 1 November 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 microRNA 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 GFPmut3b is proportional to the number of toehold switches activated (as the more switches activated, the greater the amount of GFPmut3b is produced), thus indicating the levels of hsa-miR-15b-5p present (as the more microRNA there is, the greater the number of switches activated). This part can therefore be used to quantify the levels of hsa-miR-15b-5p.

This part contains a strong RBS sequence.

NUPACK Structure Analysis

Part characterisation

Amplification of toehold switches

We generated plasmid constructs containing sequences for GFP and an upstream toehold switch that controls the production of the GFP post-transcriptionally. Our initial plasmid constructs were under the control of the constitutive promoter BBa_J23111. To ensure we had enough constructs for testing in a cell free system, we first amplified our plasmids in E. coli. During amplification we found that the constructs were toxic to E. coli. Consequently, we replaced BBa_J23111 with the inducible promoter BBa_K808000 to reduce expression of the constructs during amplification. With BBa_K808000, we successfully amplified our parts and extracted the plasmid constructs for use in a cell free system.

During our experiments with another set of toehold switch constructs (BBa_K2206003, BBa_K2206005 and BBa_K2206007), we found the same issue with construct toxicity. Therefore, we recommend that toehold switch constructs should initially be designed with non-leaky promoters for use in E. , however, we found that BBa_K808000 was too leaky for regulating these constructs.

Results

The toehold switch that controls GFP production is regulated by the presence or absence of the microRNA (miRNA) hsa-miR-15b-5p (15b-5p). We characterised the toehold switch activity in a cell free system in response to varying concentrations of 15b-5p. We tested our toehold switches with concentrations ranging from 90fM to 900nM, with concentrations increasing by an order of magnitude for each test. We characterised toehold switch activity at 30 °C and recorded fluorescence intensity every 10 minutes for 10 hours.

When testing the switch with 90nM of miRNA we found that one of our replicates was invalid and as such we omitted that set of results from our analysis.

Set time to:

We found our toehold switch required concentrations in the range of 9-90nM to be significantly activated when 1μl of 15-5p was added. We also found that fluorescence intensity increased with miRNA concentration.

-ve control is just the cell free extract and the -ve mix is the cell free extract + arabinose + DNA without miRNA

The results showed that the sensors were activated in under an hour and that fold-changes (the fluorescence compared to the system without miRNA) were greater than 20-fold after 4 hours. The maximum fold-change achieved was 25.5-fold at 10 hours with 900nM of 15b-5p. We also found that the fold-change was lower when 90nM of 15b-5p was added, with a maximum fold-change achieved being 19-fold at 10 hours.

Specificity

To ensure that toehold switch activation did not occur in the presence of a random miRNA sequence, we also tested the system with 1 μl of 900nM hsa-miR-27b-3p (27b-3p). We observed a mean fold change of 1.25 throughout the 10 hour period that the experiment was ran for, with the fold-change never surpassing 1.33. This indicates that our toehold switches are only activated in the presence of the correct target miRNA (15b-5p).

Conclusion

We found noticeable changes in fluorescence output in response to different miRNA concentrations. Our results therefore showcase the potential that toehold switches have for miRNA quantification. For information about how we applied this, visit http://2017.igem.org/Team:CLSB-UK. Another key finding was that our toehold switch constructs were toxic to E. coli, therefore, we recommend that future toehold switches should be under the control of a non-leaky, inducible promoter to allow for use in E. coli.

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 5: / Line 5: @@
 This part codes for a toehold switch that contains a region that is complementary to the microRNA 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 GFPmut3b is proportional to the number of toehold switches activated (as the more switches activated, the greater the amount of GFPmut3b is produced), thus indicating the levels of hsa-miR-15b-5p present (as the more microRNA 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 suitable for expressing this part.
 This part contains a strong RBS sequence.
@@ Line 16: / Line 14: @@
 ===Amplification of toehold switches===
-We generated plasmid constructs containing sequences for GFP and an upstream toehold switch that controls the production of GFP post-transcriptionally. Our initial plasmid constructs were under the control of the constitutive promoter BBa_J23111. To ensure we had enough constructs for testing in a cell free system, we first amplified our plasmids in E. coli. During amplification we found that the constructs were toxic to E. coli. Consequently, we replaced BBa_J23111 with the inducible promoter BBa_K808000, to reduced expression of the constructs during amplification. With BBa_K808000, we successfully amplified our parts and extracted the plasmid constructs for use in a cell free system.
+We generated plasmid constructs containing sequences for GFP and an upstream toehold switch that controls the production of the GFP post-transcriptionally. Our initial plasmid constructs were under the control of the constitutive promoter BBa_J23111. To ensure we had enough constructs for testing in a cell free system, we first amplified our plasmids in ''E. coli''. During amplification we found that the constructs were toxic to ''E. coli''. Consequently, we replaced BBa_J23111 with the inducible promoter BBa_K808000 to reduce expression of the constructs during amplification. With BBa_K808000, we successfully amplified our parts and extracted the plasmid constructs for use in a cell free system.
+During our experiments with another set of toehold switch constructs (BBa_K2206003, BBa_K2206005 and BBa_K2206007), we found the same issue with construct toxicity. Therefore, we recommend that toehold switch constructs should initially be designed with non-leaky promoters for use in ''E. , however, we found that BBa_K808000 was too leaky for regulating these constructs.
 ===Results===
-The toehold switch that controls GFP production is regulated by the presence or absence of the microRNA (miRNA) hsa-miR-15b-5p (15b-5p). We characterised the toehold switch activity in a cell free system in response to varying concentrations of 15b-5p.
+The toehold switch that controls GFP production is regulated by the presence or absence of the microRNA (miRNA) hsa-miR-15b-5p (15b-5p). We characterised the toehold switch activity in a cell free system in response to varying concentrations of 15b-5p. We tested our toehold switches with concentrations ranging from 90fM to 900nM, with concentrations increasing by an order of magnitude for each test. We characterised toehold switch activity at 30 °C and recorded fluorescence intensity every 10 minutes for 10 hours.
-We tested our toehold switches with concentrations ranging from 90fM to 900nM, with concentrations increasing by an order of magnitude with each test. We characterised toehold switch activity at 37 °C and recorded fluorescence intensity every 10 minutes for 10 hours.
 {{CLSB-UK 17 Results 90nM Repeats}}
@@ Line 41: / Line 39: @@
 ===Specificity===
-To ensure that toehold switch activation did not occur in the presence of a random miRNA sequence, we also tested the system with 1 μl of 900nM hsa-miR-27b-3p (27b-3p). We observed a mean fold change of 1.25 throughout the 10 hours that the experiment was left to run, with the fold-change never surpassing 1.33. This indicates that our toehold switches are only activated in the presence of the correct target miRNA (15b-5p).
+{{CLSB-UK 17 Results 27b}}
+To ensure that toehold switch activation did not occur in the presence of a random miRNA sequence, we also tested the system with 1 μl of 900nM hsa-miR-27b-3p (27b-3p). We observed a mean fold change of 1.25 throughout the 10 hour period that the experiment was ran for, with the fold-change never surpassing 1.33. This indicates that our toehold switches are only activated in the presence of the correct target miRNA (15b-5p).
 ===Conclusion===
-We found noticeable changes in fluorescence output in response to different miRNA concentrations. Our results therefore showcase the potential that toehold switches have for miRNA quantification. For information about how we applied this, visit http://2017.igem.org/Team:CLSB-UK.
+We found noticeable changes in fluorescence output in response to different miRNA concentrations. Our results therefore showcase the potential that toehold switches have for miRNA quantification. For information about how we applied this, visit http://2017.igem.org/Team:CLSB-UK. Another key finding was that our toehold switch constructs were toxic to ''E. coli'', therefore, we recommend that future toehold switches should be under the control of a non-leaky, inducible promoter to allow for use in ''E. coli''.