All the parts needed for the InterLab Study were extracted from the 2015 iGEM Registry Distribution Kit.

Each promoter containing plasmid was digested with 1 μl of SpeI and 1 μl of PstI at 37°C overnight. The day after add 1 μl of phosphatase (CIP from New England Biolabs) for 2 hours at 37°C. The enzymes were then heat deactivated.

The digestion reactions were assembled in this way:

A single colony was inoculated with a sterile pipette tip in a test tube with 10 ml of LB and antibiotic (1000:1 LB to antibiotic ratio) and placed in the thermoshaker (190 rpm, 37°C. When the culture got cloudy, 40 ml of LB+antibiotic were added to reach a final volume of 50 ml. The cells were grown until an OD600 of 0.5 and then centrifuged at 4100 rpm for 10 minutes at 4 °C. The supernatant was discarded and the cells were resuspend in 5 ml of LB + antibiotic + glycerol (20% v/v). The cells were kept on ice and were promptly aliquoted into 200 μl tubes and frozen at -80°C immediately. From this protocol we obtained a 10X concentrated glycerol stock for each sample.

The glycerol stock was thaw and added into 10 ml of LB with antibiotic, giving a starting culture with an OD600 of 0.1. The sample were grown in a 50 mL conical plastic tube in the termoshaker at 37°C and were grown until an OD600 0.7. At this point 3 ml of the culture were transferred in a new tube, centrifuged it, and stored at -20°C, except if otherwise indicated.

The cells were grown from glycerol stock until an OD600 of 0.7 was reached. Total RNA was purified by using the Thermo Scientific GeneJET RNA Purification Kit, following the manufacturer`s instructions and subsequently genomic DNA was removed from the total RNA by using the Thermo Scientific RapidOut DNA Removal Kit, following the manufacturer`s instructions. RNA levels were quantified using NanoDrop 1000 and reverse transcription of cDNA from the RNA template was performed with Thermo Scientific RevertAid First Strand cDNA Synthesis Kit, following the manufacturer`s instructions. qPCR reactions were performed with BioRad CFX96 TouchTM Real-Time PCR Detection System (made in USA) and assembled as follows:

Primers were designed for the reporter gene GFP_mut3b and for the housekeeping gene idnT (D-gluconate transporter) as indicated above.

We then analyzed the raw data, calculating the relative fold expression of each GPF device compared to the housekeeping (ΔCt) and the related standard deviation:

The cells were grown from glycerol stocks as described above. Differently from before when they reached the OD of 0.7 were not frozen, but were used immediately to measure fluorescence intensity. An aliquot of 5 μl of cells was diluted in 900 μl of PBS. The instrument used was a BD FACSCanto (made in USA) set with the following parameters:

• FSC gain: 525 V
• SSC gain: 403 V
• FITC gain: 510 V
• Flow rate: LOW
• Total number of events in P2: 10000

Those parameters allowed the instrument to process 900/1500 events per second. We analyzed the raw data and plot the means of three replicates with relative standard deviations.

Miniprepped DNA was purified and extracted through phenol/chloroform extraction followed by ethanol precipitation. Reactions were set following Promega E. coli S30Extract System Technical Bulletin and were performed using Qiagen Rotor-Gene Q (made in USA). Parameters for this specific experiment were set as follows:

• Green channel gain: 0.67
• Green channel Excitation: 365±20 nm
• Green channel Emission: 510±5 nm

We analyzed the raw data and plot the means of three replicates over time: