Our RFP internal standard (IS) was developed to address the issue that copy number of the same plasmid is not consistent across cells. The inclusion of an IS RFP signal is therefore designed to allow measurement of variation in gene expression between cultures and transformant lines. The underpinning assumption is that because the IS devices are identical across each of the plasmids, the GFP fluorescence values of the test devices of interest can be reported relative to their internal RFP signal.

RFP was cloned into the non-coding region between the chloramphenicol resistance gene and the ORI. The new plasmids were tested Analysis of the internal standards involved comparing the original InterLab test device plasmid performance against the new internal standard plasmid performance.

Results show that inclusion of the IS altered the behaviour of the test devices. The IS repressed expression of GFP from all devices and affected expression rates - particularly in devices with strong promoters. Further, the IS signal varied across plasmids though the only difference was the strength of the test device. The data generated using this approach indicates competition for cellular resources between the IS and test device. As a result, measurements and conclusions regarding the strength of strong reporters should be treated with caution.

http://parts.igem.org/Part:BBa_K2797013

https://2018.igem.org/Team:Newcastle/Measurement

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