Team:ZJU-China/project s1 3.htm



Characterization of clover version 2


We first characterize clover version 2 (Part K738002) we designed in split GFP methods.


Clover version 2 is synthesized by Genscript in pETDuet-1 backbone and is a regulatory and tunable allosteric scaffold (alloscaffold). Allosteric function is realized by theophylline aptamer improved from K537009 & K411003 and scaffold function is realized by basic D0.

As we design it, when theophylline is added, MS2 aptamer will be free to combine FA tagged MS2 protein and closer with PP7 aptamer thus give out increasing light. We use fluorescence microscope (Olympus BX41), confocal microscopy and Biotek Synergy H1 Hybrid Reader to test our alloscaffold clover version 2. For Biotek Synergy H1 Hybrid Reader experiments, fluorescence intensity/ OD 630 are used to be measurement criteria for comparison of the ability of give out light in Biotek Synergy H1 Hrbrid Reader test.


1. Scaffold effect


We use fluorescence complementation to detect 3D comformational change of our alloscaffold clover version 2. Green fluorescent protein (GFP) split into two halves (FA and FB) fused to the PP7 or MS2 aptamer binding proteins was used. Cells expressing FA and FB alone or clover version 2 without the split GFPs displayed little fluorescence. However, the coexpression of clover version 2 with the split GFPs showed increased fluorescence. Thus, our alloscaffold clover version 2 served as docking sites to promote protein-protein interactions in cells.


We make a series of cotransformation containing pCJDD0, pZCCOV 2,pCJDFA,pCJDFB, pCJDFA + pCJDD0, pCJDFB + pCJDD0, pCJDFB + pZCCOV 2, pCJDFA + pCJDFB,and pCJDFA + pCJDFB + pCJDD0 & pCJDFA + pCJDFB + pZCCOV 2. At mid-log phase we low-temperature induced the E.colis by adding IPTG (0.2mM & 1mM) at 20℃. After 5 hours, strains containing clover version 2 were adding 0.5mM Theophylline (optimum concentration). Successfully results are shown as we expected that, strains expressing only RNA scaffolds almost don’t give out light, strains expressing FA+FB give out medium light and strains containing alloscaffold &FA+FB emit a powerful beam after excitation.


To our surprise, the alloscaffold clover version 2 serves as a more effective scaffold than the origin D0, whose luminescence efficiency is 124.67% and performs better than D0 by 39.5%.


(Formula: `luminescence \quad efficiency=\frac{\frac{FI}{OD(FA+FB+scaffold)-\frac{FI}{OD(FA+FB)}}}{\frac{FI}{OD(FA+FB)}}`


Click here to view results.


2. Regulate and control by Theophylline


To characterise the theophylline tuned alloscaffold clover version 2 (part K738002), we quantified their activation at different theophylline concentrations (0 mM,0.1mM, 0.2 mM, 0.3mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM,1 mM) over a period of time using fluorometry. Competent E. coli (strain BL21*DE3) cells were transformed with plasmid vectors containing the alloscaffold and were cultured until the mid-log phase of growth, then 0.2mM IPTG were added. After 3 hours, a different concentration of theophylline was added to each culture for induction. 2 hours later, we use PBS to wash the culture and prepare for following tests. The regulatory effect of the Theophylline aptamer was detected as a fluorescent response as a result of increased release of the MS2 aptamer. MS2 aptamer will be free to combine MS2 protein tagged FA and be closer with PP7 aptamer when Theophylline adding. Biotek Synergy Hybrid Reader was used to excite the cultures at 480 nm and the intensity of the emission peak was detected at 535 nm. 3 replications were taken each culture, and 7 repeated experiment.


We find that up to a certain point (0.5 mM), a positive correlation exists between the GFP production and theophylline concentration, indicating that at first MS2 aptamer and theophylline aptamer are interacting(through specific base pairing) and close, when theophylline goes in, MS2 aptamer combines FA+MS2 and interact with FB+PP7 on PP7 aptamer thus give out fluorescence light. More theophylline, more light. Click to view results here.

We process these data with SAS software GLM procedure, ANOVA results show significant variation(P-value<0.05) between 0mM theophylline and 0.5mM theophylline (optimum theophylline concentration to make clover version 2 work) effects on clover version 2. More details can be found in Results.


It turns out that our alloscaffold clover version 2 can be regulated and controlled through conformational change by theophylline. This scaffold, by theophylline management, could have a variety of functions, more than accelerate the reaction, but also tune the degree of acceleration, or even reduce the reaction rate.


Characterization of clover version 3

Although clover version 2 shows function of tunable scaffold, some problems remain, for example, in the absence of theophylline, it also shows a remarkable function of scaffold, which means theophylline aptamer’s base paring doesn’t block MS2 aptamer’s combination with MS2 significantly. And, clover version 2 doesn’t response to theophylline distinctly.

Then after evaluation, clover version 3 was designed and synthesized.

We use controlled trial to compare theophylline’s regulatory effect on clover version 2 & 3. Competent E. coli (strain BL21*DE3) cells were transformed with plasmid vectors containing pCJDFA + pCJDFB + pZCCOV 2 and pCJDFA + pCJDFB + pZCCOV 3. Cultured until the mid-log phase of growth, 0.2mM IPTG and theophylline(0mM and 0.4mM for comparison) were added, and we contrast experiment in 20℃ and 37℃. After 4 hours, we use PBS to wash the culture and carry out Hybrid Reader tests. 3 replications were taken each culture, and 8 repeated experiment. Results show that clover version 3 works more prominently than clover version 2. Whatever the temperature is, clover version 3 responses to theophylline more sharply than clover version 2, and when theophylline is absent, clover version 3 shows less scaffold function, which means theophylline does reopen the block of MS2 aptamer.