For this study, three devices needed to be constructed consisting of a constitutive Anderson promoter with low (J23117), medium (J23106) and high (J23101) promoter strengths expressing GFP (I13504) as reporter. Using the Biobrick distribution, the required promoters and GFP were cloned in E.coli top10 cells, restricted and ligated to afford the final devices. Fluorescence was measured from overnight cultures using FACS (fluorescence-activated cell sorting) to compare the individual promoter strengths in relative units.

To measure fluorescence, the cells were excited at a wavelength of 488 nm and emitted light was measured at 530 ± 30 nm and a total of 30’000 events were recorded. The data was shown in clouds of fluorescence on the x axis and fluorescent side scattering on the y axis.

Negative and positive control

Plasmids containing only the promoter without GFP were used as negative controls to calibrate the FACS machine. The Biobrick I20270 (containing J23151 as constitutive promoter, obtained from the distribution) was used as a positive control. x label: fluorescence (relative units); y label: FSC fluorescence side scattering (cell size)

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High promoter strength

J23101_I13504 was grown in biological triplicate (hi clone 1,2,3) and was measured in technical triplicate (a,b,c). x label: fluorescence (relative units); y label: FSC fluorescence side scattering (cell size)

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Medium promoter strength

J23106_I13504 was grown in biological triplicate (med clone 1,2,3) and was measured in technical triplicate (a,b,c). x label: fluorescence (relative units); y label: FSC fluorescence side scattering (cell size)

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Low promoter strength

J23106_I13504 was grown in biological triplicate (low clone 1,2,3) and was measured in technical triplicate (a,b,c). x label: fluorescence; y label: FSC fluorescence side scattering (cell size)

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To process our data, the relative fluorescence of the corresponding negative controls was subtracted from the measured samples to get relative data for our three constructs.

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As expected, high, medium and low levels of fluorescence were measured in accordance with the promoters used in our constructs. The weak promoter J23117 however was much weaker than was expected with only approximately 15 relative units higher than the negative controls. In contrast, the medium and high promoters lead to a significant increase in measured fluorescence. J23106 is approximately 70 times stronger than J23117, and J23101 approximately 170 times stronger than J23117. The high promoter J23101 thus is approximately 2.3 times stronger than the medium promoter J23106.

Statistical analysis of our data using standard deviations showed that differences among the biological triplicates are larger than instrument fluctuations (biological SD is roughly 5 to 15 times larger than techical SD). Thus, repeated measurement of the same sample generates a statistically more significant result than measuring several biological samples once. Based on standard deviations of roughly 10% for biological triplicates and 5% for technical triplicates, we recommend to increase the data set in order to improve statistical relevance.

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