Team:ZJU-China/project s1 5.htm

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Latest revision as of 22:42, 26 October 2012

Results

1. Scaffold effect

Fig.15 Different RNA scaffold’s effect on split GFP showing by fluorescence microscopy. The BL21*DE3 of the E. coli were transformed with pCJDFA+pCJDFB, pCJDFA+pCJDFB + pCJDD0, and pCJDFA+pCJDFB + pZCCOV 2 (0.5 mM theophylline adding). As expected, strains without RNA scaffold did not fluoresce. Upon the existence of RNA scaffold, many of the cells emitted fluorescence indicating a substantial amount of split GFP combination is permitted because of the function of RNA scaffold. The brightfield images in the right column depict all bacterial cells. The GFP images in the left column depict bacterial cells which emitted fluorescence.

Fig.16 Biotek Synergy H1 Hybrid Reader controlled experiments. The BL21*DE3 of the E. coli were transformed with figure showing plasmids. (0.5 mM theophylline was adding in strains containing clover 2).

`luminescence \quad efficiency \quad of \quad clover 2=\frac{\frac{FI}{OD(FA+FB+clover 2)}-\frac{FI}{OD(FA+FB)}}{\frac{FI}{OD(FA+FB)}}=\frac{53425-23779}{23779}=125\%`

`luminescence \quad efficiency \quad of \quad D0=\frac{\frac{FI}{OD(FA+FB+clover 2)}-\frac{FI}{OD(FA+FB)}}{\frac{FI}{OD(FA+FB)}}=\frac{38288-23779}{23779}=61\%`

The original intention of our designing RNA scaffold clover 2 is to create a regulatory scaffold which can tune its conformation thus have various functions. To our surprise, clover version 2, when adding optimal Theophylline concentration 0.5mM, happens to be a more powerful scaffold which helps two halves of GFP’s combination and give out light strongly.

One possible reason is in clover version 2, distance between MS2 aptamer and PP7 aptamer is closer than in D0 (showing in 04 S1 Rboscaffold Fig.4 and Fig.6), so that when binding phage coat proteins, FA and FB on clover version 2 were set closer than on D0. We submit the inference that when RNA scaffold binds enzymes, clover version 2 draws two enzymes nearer than D0 thus has more ability to accelerate the enzymatic reaction.

2. Regulate and control by Theophylline

When the concentration of Theophylline is in the range from 0mM to 0.5mM, the concentration of Theophylline and the resulting fluorescence intensity are directly proportional.

Theophylline concentration beyond certain extent will be hazardous to cells and how it affects cells depends on strain type. The study by NYMU Taipei 2010 alerted adding more than 4mM of Theophylline would cause E. coli to die. In our experiments, we find that after adding more than 0.5mM, the Theophylline spectrum curve would be invalid. As a result, we pick up data with concentrations below 0.5mM to analyze as the E. coli cell would be unstable or the regulation of the Theophylline aptamer would not be accurate. See the original data.

Fig.17 7 tests of fluorescence/ OD change over theophylline concentration. There’s evident positive correlation in between.

Then we build several SAS models to analyze data with SAS software GLM procedure between 0-0.5mM Theophylline concentrations of treatments, choosing” clover version 2: different treatments versus blocks” test 5-7 to run a SAS model.

ANOVA result P-value shows that Theophylline concentrations have significant impact on fluorescence intensity of clover version 2 and almost no impact on D0. That is to say, our designed RNA scaffold clover version 2 can be regulated and controlled by Theophylline within 0-0.5mM not for random errors or common phenomenon in RNA scaffolds.

If you want more details about SAS source programs and software computational results, please click here [code].

3. Clover version 3 versus 2

Fig.18 Biotek Synergy H1 Hybrid Reader controlled experiments. The BL21*DE3 of the E. coli were transformed with figure showing plasmids. Expression was under 37℃. (0.4 mM theophylline was adding for contrast with theophylline absence.) 0.4mM theophylline increases clover version 2’s luminescence efficiency by 30.77% and clover version 3 by 57.23%. Also, when theophylline is absent, clover version 3 shows less fluorescence intensity. It turns out that clover version 3 response to theophylline more sensitively, thus more efficient to regulate.

Fig.19 Biotek Synergy H1 Hybrid Reader controlled experiments. The BL21*DE3 of the E. coli were transformed with figure showing plasmids. Expression was under 20℃. (0.4 mM theophylline was adding for contrast with theophylline absence.) 0.4mM theophylline increases clover version 2’s luminescence efficiency by 13.27% and clover version 3 by 41.31%. Also, when theophylline is absent, clover version 3 shows less fluorescence intensity. It turns out that clover version 3 responses to theophylline more sensitively, thus more efficient to regulate.

Results show that our improvement of clover version 2 succeeds. Clover version 3 behaves more responsive to theophylline than clover version 2 thus more controllable and tunable. See original data. And in clover version 3, primary scaffold function in the absence of theophylline is inconspicuous, which foresees in our future design and optimization, the alloscaffold’s behavior could reach “0-1” step response (when theophylline is absent, alloscaffold almost doesn’t work; when adding theophylline, alloscaffold works as scaffold significantly.)