Team:TU Munich/Parts

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Malachitegreen-Binding Aptamer - BBa_K494000


Methods to visualize nucleic acids via fluorescence are rare, partly due to the size of fluorescent reporters. Thus, we present the malachitegreen-binding aptamer to the partsregistry. By adding only 37 bp, fluorescent determination of specific nucleic acids becomes possible allowing evalutation of PoPS devices via in vitro transcription. Binding of triphenyl dye malachitegreen to the aptamer increases fluorescence by 2360-fold. This leads to an significant increase and a shift in absorbance from 618 to 630 nm. With an emission maximum at 652 nm, aptamer-bound malachitegreen fluoresces at longer wavelength than most dyes and does not interfere with those. [1] We provide this part for efficient in vitro evaluation of PoPS devices in general and switches based on our concept in particular.

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The malachitegreen-binding aptamer has been successfully used in screening systems being both robust and easy to produce. Aptamers provide specifities in the range of antibodies and can be evolved to target small molecules and proteins. [1]
Since malachitegreen is a membrane permeable dye, its uses are not limited to in vitro measurements. The malachite green aptamer can be used to tag and follow any RNA, including messengar and small RNAs to study questions about their metabolism and biological functions.[1] Aside from the application as a mere reporter, the malachitegreen-binding aptamer has already been utilized to build up modular sensors which can together with another RNA-binding domain sense and report small molecules like ATP for example. This new detection method seems to provide promising future applications and sensors. Since the principle of modularizing fits well into our concept of building networks, we like to provide this part to allow further engineering considering in vitro sensing systems. [1]

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Plasmids

In general we want to provide a new principle of gene regulation which can be further developed, tested and optimizted by everybody. Therefore we focus on providing the parts needed for verification and testing of new individual switches. We provide a plasmid which can be used for further cloning, a positive control to test the general functionality and the constructs we characterized for comparison.

BBa_K494001

We designed a new screening systems based on the non-functional pSB1A10 plasmid. We improved its features for ‘’in vivo’’ characterization of PoPS devices using fluorescent reporters . The plasmid still contains the Pbad arabinose-inducible induction system as a tunable input and eGFP as an internal standard for induction. However, we altered the reporter protein to mCherry. Furthermore we adjusted the BioBrick cloning site to allow cloning of additional parts independent from the Input/Output measurement. This screening plasmid is designed to be used with a second Arabinose inducible promoter <partinfo>I13453</partinfo> which is not included in this part.

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The improved screening system is optimized for the evaluation of PoPS devices in fluorescence measurements. RFP which was known to contain an RNase restriction site was replaced by mCherry which combines good expression yield, short maturation times and an acceptable and well-characterized quantum yield. A unique challenge for the characterization of our switches is the expression of a corresponding signal independent of the input/output measurement. Thus, we moved the BioBrick cloning site resulting in the fluorescent reporter being inside the cloning site and giving the possibility to clone independent parts behind the reporter protein. To fully function our screening plasmid need the arabinose inducible promoter BBa_I13453 in front of the PoPS device to screen. Using a second arabinose inducible promoter, we were able to keep eGFP as an internal standard for the tunable input via the Pbad arabinose-inducible induction system. The two identical promoters ensure the same rate of induction for eGFP and the tested PoPS-device. Thus, obtaining comparable screening results is easy. Unfortunately, this design implicates a minor disadvantage. Two cloning steps are needed to gain an functional construct for testing any PoPS device.

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Screening system: Positive Control BBa_K494002

TUM2010 PicPosControl3cInd.png

Here we present a ready to use composite part consisting of our screening system pSB1A10mod <partinfo>BBa_K494001</partinfo>, the PBad Promotor <partinfo>BBa_I13453</partinfo> and the reporter protein mCherry <partinfo>BBa_J06702</partinfo> including RBS and double Terminator <partinfo>BBa_B0015</partinfo>. This part belongs to the screening system and is intended as positive control.

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Emission spectra of induced pSB1A10mod positive control BBa_K494002 ; A: eGFP fluorescence ex: 501 nm, B: mCherry fluorescence ex: 587 nm

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BBa_K494003

With His-Term/Signal

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BBa_K494004

With Trp-Term/Signal

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Falsification

pSB1A10

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References

[1] Babendure, J.R., S.R. Adams, and R.Y. Tsien, Aptamers switch on fluorescence of triphenylmethane dyes. J. Am. Chem. Soc, 2003. 125(48): p. 14716-14717. [2] Stead, S.L., et al., An RNA-Aptamer-Based Assay for the Detection and Analysis of Malachite Green and Leucomalachite Green Residues in Fish Tissue. Analytical chemistry. 82(7): p. 2652-2660. [3] Stojanovic, M.N. and D.M. Kolpashchikov, Modular aptameric sensors. J. Am. Chem. Soc, 2004. 126(30): p. 9266-9270. [4] https://2008.igem.org/Team:Heidelberg [5] Smolke and so on.... [6] http://en.wikipedia.org/wiki/Logic_gate#Symbols

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