Parts Overview

Submitted Parts

Parts Description

Malachite Green 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 malachite green-binding aptamer to the partsregistry. By adding only 38 bp, fluorescent determination of specific nucleic acids becomes possible allowing evalutation of PoPS-based devices via in vitro transcription.^[1] Binding of triphenyl dye malachite green 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 malachite green fluoresces at longer wavelength than most dyes and does not interfere with those.^[2] We provide this part for efficient in vitro evaluation of PoPS-based devices in general and switches based on our concept in particular.

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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.

Screening system: Backbone 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-based 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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Screening System: Positive Control BBa_K494002

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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Screening System: Negative Control BBa_K494003

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

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Emission spectra of induced pSB1A10mod HisTerm BBa_K494004 ; A: eGFP fluorescence ex: 501 nm, B: mCherry fluorescence ex: 587 nm

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Screening System: Control BBa_K494004

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

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Emission spectra of induced pSB1A10mod TrpTerm BBa_K494004 ; A: eGFP fluorescence ex: 501 nm, B: mCherry fluorescence ex: 587 nm

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Screening System: BBa_K494005

Screening System: BBa_K494006

Falsified Parts

pSB1A10

The Screening Plasmid pSB1A10 is intended for the characterization of an Input/Output for a PoPS based device on the basis of two fluorescent proteins. While the first fluorescent protein, eGFP quantifies the input from an arabinose induced promoter, the second reporter, RFP detects the output.

However, our experiments designed for testing toggle switches revealed the part to be non-functional. In the current available form, RFP fluorescence is not induced even when the PoPS device does not interfere with the Polymerase.

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Kinetic measurement of pSB1A10(induced & uninduced). GFP fluorescence ex: 501 nm, RFP fluorescence ex: 584 nm

Although induction with arabinose worked fine indicated by GFP fluorescence, we could not detect any RFP output. The kinetic measurment on the left shows pSB1A10 postive control with a nonsense sequence inserted into the BioBrick site between the two fluorescent proteins GFP and RFP. Nevertheless induction does not trigger any RFP expression. Therefore no output signal is created in our experimantal setup. This fact renders the part as it is unsuitable for testing PoPS-based devices. We first assumed degradation of RFP-mRNA which is known to have an RNase site might be responsible. But comparison with our new screening system reveals a transcriptional problem to be most likely as replacing RFP did not improve the construct. Though, introducing a second arabinose promoter finally resulted in the desired output signal.

In order to create a functional screening device for measurements we suggest cloning an additional PBad promoter <partinfo>BBa_I13453</partinfo> in front of the part to be tested. Thus, eGFP is still able to function as an internal standard for expression rate via the Pbad arabinose-inducible induction. Therefore comparable results for PoPS-based devices are achievable.

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References

[1] Grate, D. and C. Wilson, Laser-mediated, site-specific inactivation of RNA transcripts. Proceedings of the National Academy of Sciences of the United States of America, 1999. 96(11): p. 6131.

[2] 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.

[3] Rowe, W., M. Platt, and P.J.R. Day, Advances and perspectives in aptamer arrays. Integrative Biology, 2009. 1(1): p. 53-58.

[4] Stojanovic, M.N. and D.M. Kolpashchikov, Modular aptameric sensors. J. Am. Chem. Soc, 2004. 126(30): p. 9266-9270. [5] Smolke and so on.... [6] http://en.wikipedia.org/wiki/Logic_gate#Symbols