Difference between revisions of "Part:BBa K4061090"
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| − | This composite part was made to characterise the efficiency of antisense | + | This composite part was made to characterise the efficiency of antisense RNA as designed in BBa_K4061041 in reduction of noise. We also regulated the antisense RNA under a PompF promoter (BBa_K4061000) to analyse the promoter's function. Rationale being that in the condition when the OmpF promoter should be active, the antisense RNA will be more in number, therefore reducing fluorescence intensity. This construct gave us dual information- about antisense RNA efficiency and the promoter function. |
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Latest revision as of 14:12, 19 October 2021
eGFP antisense characterisation
This composite part was made to characterise the efficiency of antisense RNA as designed in BBa_K4061041 in reduction of noise. We also regulated the antisense RNA under a PompF promoter (BBa_K4061000) to analyse the promoter's function. Rationale being that in the condition when the OmpF promoter should be active, the antisense RNA will be more in number, therefore reducing fluorescence intensity. This construct gave us dual information- about antisense RNA efficiency and the promoter function.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1325
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 and the promoter.
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 1, the antisense RNA when inserted along with constitutively expressed eGFP significantly reduces the fluorescent intensity. However, it appears that the fluorescence intensity of eGFP-antisense construct is comparable across the entire range of medium osmolarities. This indicates that PompF functions equally over different ranges of osmolarity. This contradicts our hypothesis since the antisense fragment would theoretically not be expressed at higher medium osmolarity. In order to characterise OmpF promoter's activity, we conducted several experiments as documented on BBa_K4061000 as well and the declining trend as the osmolarity increases seems apparent. Therefore, this result is likely due to limitations of the assay and not the promoter's function. Nonetheless, our major takeaways from this experiment was that the antisense RNA is very effective in reducing noise from eGFP molecules.
