Part:BBa_K4061041
Antinsense RNA against Constitutive eGFP
(Not to be confused with BBa_K4061040 which is similar antisense RNA just optimised for OmpC promoter)
This is an antisense RNA encoded to downregulate Constitutively expressed (BBa_J23100) eGFP fluorophore. This RNA will be a part of the composite part (BBa_K4061090). This RNA was used to check if the antisense RNA design spanning the protein CDS, 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 eGFP. Teams working with BBa_J23100 BBa_B0034 and BBa_K4061002 (Constitutive promoter, Strong RBS and eGFP) 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 the antisense RNA (BBa_K4061041) in suppression of eGFP production- seen as reduction in the fluorescence intensity. We regulated the antisense RNA under the OmpF promoter (BBa_K4061000). This could provide insights into efficiency of the antisense RNA.
Experiments
We built a construct with a constitutively expressed (BBa_J23100) eGFP reporter in tandem with the OmpF promoter regulated antisense RNA against eGFP. This construct was tested against just constitutively expressed eGFP. 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.
Results and Discussion
As seen in the graph, the antisense RNA when inserted along with constitutively expressed eGFP significantly reduces the fluorescent intensity. The intensity of fluorescence reduced 4-8 times in the presence of the antisense RNA.
The Hfq binding domain on the antisense molecule must be offering stability to the antisense RNA, however we are yet to characterise the activity and efficiency of the binding domain. We encourage teams to eliminate the domain and conduct the plate-reading for fluorescence intensity with constructs with and without the hfq binding domain on the antisense RNA.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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