Part:pSB1A10:Experience

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Falsification

The [http://2010.igem.org/Team:TU_Munich TUM iGEM Team 2010] tryed to use the part for screening PoPS-devicec. It did not work at all. 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 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.

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 BBa_K494001reveals 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 BBa_I13453 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. The BBa_K494001 screening plasmid based on pSB1A10 is also available.

User Reviews

UNIQ31f613763b9ca301-partinfo-00000003-QINU

••••• Antiquity

This review comes from the old result system and indicates that this part worked in some test.

UNIQ31f613763b9ca301-partinfo-00000005-QINU