Difference between revisions of "Part:BBa K2206012"

Latest revision as of 13:25, 8 October 2021

Toehold switch for hsa-miR-15b-5p (second series)

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.

The trigger sequence can be split into different RNA molecules. Colocalization of the split trigger parts results in a complete trigger RNA that is capable of activating the toehold switch.

BBa_K2206013 and hsa-miR-15b-5p contain roughly half of the trigger sequence each. Colocalization of the split trigger RNA sequences results in a functional trigger RNA, capable of activating the toehold switch. The formation of the duplex occurs through a highly specific binding step which results in single-base mismatch specificity. This part codes for a toehold switch that has a region complementary to the complete trigger RNA sequence formed of BBa_K2206013 and hsa-miR-15b-5p Therefore, this part can be used to regulate the expression of any protein in response to presence of both BBa_K2206013 and hsa-miR-15b-5p.

For more information, see the section on second series of toehold switches on the design page of CLSB-UK's 2017 wiki - http://2017.igem.org/Team:CLSB-UK/Design.

We recommend using this part to regulate the expression of a reporter protein. This allows for quantification of hsa-miR-15b-5p. Our simulations show that this part would be better suited for hsa-miR-15b-5p quantification than our original toehold switch BBa_K2206000 due to its increased specificity and on state activity.

We found that the transcripts produced by our other toehold switch parts were toxic to E.coli. We therefore recommend using this part with a non-leaky promoter. For reference, we found the part BBa_K808000 was too leaky when regulating the expression of one of our previous toehold switches.

This part contains a strong RBS sequence.

NUPACK Structure Analysis

Information contributed by City of London UK (2021)

This toehold switch was characterized in silico using the ToeholdTools project that our team developed. See https://github.com/lkn849/thtools for more information.

Metadata:

• Group: City of London UK 2021
• Author: Lucas Ng
• Summary: Used our software ToeholdTools to investigate the target miRNA specificity and activation of this part.

Raw data:

• Media:BBa_K2206012_thtest.txt
• Media:BBa_K2206012_crt.txt

This contribution was autogenerated by the script contrib.py, available at https://github.com/lkn849/thtools/tree/master/registry.

This switch was designed to detect the miRNA hsa-miR-15b-5p at a temperature of 37°C. We tested it against every mature Homo sapiens miRNA in miRBase and our analysis shows that at this temperature it is best used to detect hsa-miR-3940-5p.

With hsa-miR-3940-5p at 37°C, the switch has a specificity of 21 ± 100 % and an activation of 9 ± 6 %. These values represent 95% confidence limits (z=1.96).

The temperature–activation–specificity relationship is shown here. CRT is an acronym for CelsiusRangeTest, the class in our Python library responsible for the following graph:

Error bars represent the standard deviation. The line of best fit was calculated using a univariate cubic spline weighted inverse to each point's standard error.

Caveats:

• As per the above, we cannot confirm that this switch accurately detects the desired miRNA sequence.
• The miRNA most targeted by this switch heavily fluctuates based on temperature.Therefore, we cannot confirm the reliability of this switch.

We do not recommend this part for future usage.

Sequence and Features