In the interest of promoting reproducible science, and enabling others to build off of our work, we have included both the precise methods used as well as the raw data (in csv format) for every experiment mentioned on our wiki. Experiments are split up by the page on which they are mentioned. For experiments performed with flow cytometry, .fcs files for both measurements and calibration beads (Spherotek RCP-30-5A, lot number: AB01) are included in the file. In all cases at least 10,000 single cell measurements were collected for flow cytometry data. Whenever used, the term biological replicates refers to a distinct colony from a transformation of the same miniprep. Whenever plate reader fluorescence data is provided, it is given in arbitrary units of fluorescence normalized to cell density (recorded in either Absorbance 600 or Absorbance 700).

• Circuits used were on psb1C3
• The E. coli strain used was JS006 (K-12 derivative with ΔAraCΔLacI)
• The media used was M9 minimal media with 0.4% Casamino Acids and 0.4% Glucose (see protocols)
• Antibiotics in minimal media were used in the following concentrations [Chloramphenicol: 7.5mg/mL, Kanamycin: 35mg/mL]
• Transformations were plated on selective LB plates with either 25mg/mL Chloramphenicol, 50mg/mL Kanamycin or both
• 96 well plates used with the microplate reader (Synergy H1 [Biotek]) were Black Griener Fluotrack 200 flat bottom plates with the matching lid
• Microplate reader was set to linear shaking at max speed and gradient temperatures were used to prevent condensation
• Excitation/emission used for plate reader experiments was 565/593
• The channel used for flow cytometry experiments was Fl2 (S3e cell sorter [Biorad]).

Figure 2: In both cases colonies were picked into 200µL of media into a 96 well plate and cultures were grown at 29C until cultures showed OD600s consistent with mid-log growth. Cultures were then diluted to an OD600 of ~.05 and then subjected to their conditions. For A, this was a 37C for the entire period, whereas for B this was 37C from 160 minutes to 280 minutes and 29C elsewhere. Data

Figure 3: In both cases colonies were picked into 200µL of media into a 96 well plate and cultures were grown at 29C until cultures showed OD700s consistent with mid-log growth. Cultures were then diluted to an OD600 of ~.05 and then subjected to their conditions. For A, this was a 37C for the entire period, whereas for B this was 37C from 140 minutes to 280 minutes and 29C elsewhere. Data

Figure 4: In both cases colonies were picked into 200µL of media into a 96 well plate and cultures were grown at 29C until cultures showed OD600s consistent with mid-log growth. Cultures were then diluted to an OD600 of ~.05 and then grown at 37C for 1100 minutes. Data

Figure 5: In all 3 cases colonies were picked into 200µL of media into a 96 well plate and cultures were grown at 30C until cultures showed OD600s consistent with mid-log growth. Cultures were then diluted to an OD600 of ~.05 and then grown at 39C until OD600 plateau was reached. Data

mScarlet-I RBS characterization: Circuits composed of J23107 B0034/B0032 mScarlet-I B0015 as well as a J23107 B0034 LacI B0015 control were cloned via 3G assembly onto psb1C3. 3 colonies of each circuit were picked into LB media, grown for 3 hours at 37C, diluted to OD600 0.1 and grown 4 hours. Then circuits were measured on UVAs plate reader (with emission/excitation 565/593 and absorbance 600). Data was then normalized with respect to cell density. Data

Frozen vs Non Frozen cell tests: 3 colonies of cells with BBa_K2333428 (pTet mScarlet-I pdtA) were picked into 4mL of M9 media and incubate at 37C overnight. In the morning, cells were diluted 1:100 and grown for 3 hours at 37C. Cells were diluted to a final OD600 of ~.05 in 5mL of 37C M9 and ATc was added to a final concentration of 50ng/mL. Cells were grown at 37C and 200µL time points were taken every 20 minutes (t0= immediately following inducer being added) onto ice. 50µL was filtered into ice cold PBS and measured on the FACSed immediately, while the remaining 150µL had glycerol added to a final concentration of 15%, and were then flash frozen in liquid nitrogen. Experiment was continued for 160 minutes to ensure cells remained in midlog growth. Frozen samples were stored at -80C for 3 days and then thawed on ice, 1:10 diluted into ice cold PBS and then FACSed. Data

All Sanger sequencing was performed by Epoch Life Sciences. All constructs submitted to the registry as well as many non submitted constructs were sequenced. A key for William and Mary circuit names as well as .ab1s can be found in the two part file here and here.