Part:BBa_K4061095
mRFP1 antisense characterisation
This composite part was made to characterise the efficiency of antisense RNA as designed in BBa_K4061046 in reduction of noise from mRFP1 reporter. We also regulated the antisense RNA under a PompC promoter (BBa_R0082) to analyse the promoter's function. Rationale being that in the condition when the OmpC promoter should be active (high osmolarity), 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.
Contribution: HKUST 2021
Summary
We observed the efficiency of the 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 and the promoter.
Experiments
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.
Results and Discussion
As seen in the graph, the antisense RNA when inserted along with constitutively expressed mRFP1 significantly reduces the fluorescent intensity. The difference between the fluorescence intensity is taken as a measure of the promoter. Seen on the scatter plot, the difference between the fluorescence intensity with and without the antisense RNA is significantly higher in higher concentration of medium. This supports the hypothesis that OmpC promoter functions best at high concentrations. However, at even higher concentrations of greater than 0.256M, the difference dropped. This probably is because of cells experiencing high osmotic stress at this concentration
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1085 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1077
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 616
Illegal AgeI site found at 728 - 1000COMPATIBLE WITH RFC[1000]
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