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Team:TU Munich/Parts
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We also add an malachitegreen-binding aptamer to the partsregistry which allows evalutation of terminators 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. [[Team:TU_Munich/Parts#ref1|[1]]] We provide this part for efficient ''in vitro'' evaluation of terminators in general and switches based on our concept in particular. | We also add an malachitegreen-binding aptamer to the partsregistry which allows evalutation of terminators 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. [[Team:TU_Munich/Parts#ref1|[1]]] We provide this part for efficient ''in vitro'' evaluation of terminators in general and switches based on our concept in particular. | ||
Revision as of 17:31, 24 October 2010
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New PartsSingle PartsMalachitegreen-Binding Aptamer - BBa_K494000
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] PlasmidsIn 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_K494001New, 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 ?? ?? ?? BBa_K494002Positive control BBa_K494003With His-Term/Signal BBa_K494004With Trp-Term/Signal FalsificationpSB1A10
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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