Part:BBa_K4061046
Antisense RNA to constitutive mRFP1
Not to be confused with BBa_K4061045 which is antisense RNA modified to downregulate mRFP1 under OmpF promoter.
This antisense RNA is optimised for mRFP1 under constitutive promoter. This mRNA was used to check if the antisense RNA design spanning the protein CDS of mRFP1, RBS and the -10 region of promoter was compatible to reduce noise from unwanted fluorophore. With our experiments, we can be sure that this antisense RNA is very efficient in reduction of noise from Constitutively expressed mRFP1. Teams working with BBa_J23100 BBa_B0034 and BBa_E1010 (Constitutive promoter, Strong RBS and mRFP1) could utilise this antisense molecule to suppress noise from the circuit. An additional use is if teams wish to characterise a promoter's activity, they could regulate this antisense RNA under that promoter and observe the reduction in fluorescence intensity. This can be an indicator of promoter's function.
Contribution: HKUST 2021
Summary
We observed the efficiency of this antisense RNA (BBa_K4061046) in suppression of mRFP1 production- seen as reduction in the fluorescence intensity. We regulated the antisense RNA under the OmpC promoter (BBa_R0082). This could provide insights into efficiency of the antisense RNA.
Experiments
In order to analyse the efficiency of this antisense RNA, we regulated it under the OmpC promoter which is known to upregulate at higher concentrations. We built a construct with a constitutively expressed (BBa_J23100) mRFP1 reporter in tandem with the OmpC promoter regulated antisense RNA against mRFP1. This construct was tested against just constitutively expressed mRFP1. Cells with these constructs were grown in LB medium with varying concentrations of NaCl. Fluorescence intensities from both cultures were measured using a fluorescent spectrophotometer. The difference between the fluorescent intensities were taken as a measure of antisense RNA efficiency.
Results and Discussion
As seen in the graph, the antisense RNA when inserted along with constitutively expressed mRFP1 significantly reduces the fluorescence intensity. The intensity of fluorescence reduced up to 3 times in the presence of the antisense RNA, especially in the most favourable conditions for the OmpC promoter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 57
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 49
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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