Team:TU Munich/Parts

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The introduction of fluorescent reporter  proteins into molecular biology opened endless possibilities to visualize and study biological systems. Reporter proteins can be fused to proteins of interest  to highlight expression and cellular location. Methods to visualize nucleic acids are rare, partly due to the size of fluorescent reporters. However, the malachite aptamer with only 37 bp is able to increase the fluorescence of the triphenyl dye malachitgreen by 2360fold upon binding. Thus, fluorescent determination of specific nucleic acids becomes possible.  
The introduction of fluorescent reporter  proteins into molecular biology opened endless possibilities to visualize and study biological systems. Reporter proteins can be fused to proteins of interest  to highlight expression and cellular location. Methods to visualize nucleic acids are rare, partly due to the size of fluorescent reporters. However, the malachite aptamer with only 37 bp is able to increase the fluorescence of the triphenyl dye malachitgreen by 2360fold upon binding. Thus, fluorescent determination of specific nucleic acids becomes possible.  
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. [[Team:TU_Munich/Parts#ref2|&#91;1&#93;]] <br>
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. [[Team:TU_Munich/Parts#ref2|&#91;1&#93;]] <br>
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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 messenger, ribosomal and mircro RNAs to study questions about their metabolism, trafficking and functions.[[Team:TU_Munich/Parts#ref2|&#91;1&#93;]] 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. [[Team:TU_Munich/Partst#ref3|&#91;1&#93;]]
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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.[[Team:TU_Munich/Parts#ref2|&#91;1&#93;]] 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. [[Team:TU_Munich/Partst#ref3|&#91;1&#93;]]
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Revision as of 18:00, 24 October 2010

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Malachitegreen-Binding Aptamer - BBa_K494000


We also add an malachitegreen-binding aptamer to the partsregistry which allows evalutation of PoPS-devices using in vitro transcription. Upon binding of malachitegreen to the aptamer the fluorescence increases 2360-fold leading to an significant increase over the whole transcription time 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 terminators in general and switches based on our concept in particular.

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The introduction of fluorescent reporter proteins into molecular biology opened endless possibilities to visualize and study biological systems. Reporter proteins can be fused to proteins of interest to highlight expression and cellular location. Methods to visualize nucleic acids are rare, partly due to the size of fluorescent reporters. However, the malachite aptamer with only 37 bp is able to increase the fluorescence of the triphenyl dye malachitgreen by 2360fold upon binding. Thus, fluorescent determination of specific nucleic acids becomes possible. 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

New, better pSB1A10 We recloned pSB1A10 to improve its features as a measuring plasmid to evaluate terminators in vivo using fluorescent proteins as reporters. RFP which was known to contain an RNase restriction site was exchanged against mCherry which combines good expression yield, short maturation times and an acceptable and well-characterized quantum yield. For easy introduction of the terminator to be evaluated, we ?? ?? ??

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BBa_K494002

Positive control

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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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