Dataset for supporting a modular autoinduction device for control of gene expression in Bacillus subtilis

Modular and tuneable genetic tools for Metabolic Engineering fuels the development of chassis for the efficient production of biocompounds at industrial scale. We have constructed an autoinduction device for gene expression in Bacillus subtilis based on the LuxR/I quorum sensing system [1]. Here, we present raw and processed data regarding to B. subtilis growth measured as OD600, performed in three different scales: microcultivation on 96-well plates (200 µL), test tubes (12 mL), and Erlenmeyer flasks (50 mL). We also present raw and processed data on gene expression measured as GFP fluorescence (485/535 nm), luminescence and riboflavin production. Measurements were performed on a microplate reader Tecan 200 PRO (iControl software) and on spectrophotometer (Thermo Fisher Scientific GENESYS 10S UV-Vis). Processed data are presented as product/OD600, maximum and minimum promoter activity, fold of induction, and the induction OD600.


a b s t r a c t
Modular and tuneable genetic tools for Metabolic Engineering fuels the development of chassis for the efficient production of biocompounds at industrial scale. We have constructed an autoinduction device for gene expression in Bacillus subtilis based on the LuxR/I quorum sensing system [1] . Here, we present raw and processed data regarding to B. subtilis growth measured as OD 600 , performed in three different scales: microcultivation on 96-well plates (200 μL), test tubes (12 mL), and Erlenmeyer flasks (50 mL). We also present raw and processed data on gene expression measured as GFP fluorescence (485/535 nm), luminescence and riboflavin production. Measurements were performed on a microplate reader Tecan 200 PRO (iControl software) and on spectrophotometer (Thermo Fisher Scientific GENESYS 10S UV-Vis). Processed data are presented as product/OD 600 , maximum and minimum promoter activity, fold of induction, and the induction OD 600 .

Value of the Data
• These data show how our autoinduction devices perform over single copy target genes in the bacterial chromosome. • These data support further progress in engineering B. subtilis as an efficient chassis for industrial bioprocesses. • These data will be useful for other researchers willing to use and/or further develop the autoinduction devices designed for B. subtilis . • These data could help other researchers to develop similar devices for other bacterial species.

Data Description
This article presents the dataset for supporting analysis of gene expression in B. subtilis as well as ways to assess the optimization of bacterial promoters. We present a modular autoinduc-tion device for control of gene expression at single copy in the genome. We make data available on simultaneous measurement of growth, and bioluminescence or fluorescence directly in a microplate reader. These data are part of the supplementary information for the paper "A modular autoinduction device for control of gene expression in Bacillus subtilis" [1] .
The Supplementary Data 1 presents culture optical density (OD 600 ) and luminescence raw data measured during growth in a 96-well microplate. The first worksheet handles OD 600 data: raw data (biological triplicate), processed data discounting blank, mean and standard deviation. The second worksheet handles Luminescence data: raw data (biological triplicate), normalized luminescence to OD 600 discounting blank, replace values below 0 by 0, mean and standard deviation. The third worksheet shows processed data for fold change, minimum and maximum promoter activities, and standard deviations. The fourth worksheet shows the AHL (N-(3-Oxohexanoyl)-L-homoserine lactone)-dependent induction of gene expression test, in which AHL was added to the culture medium in different concentrations, the engineered strains luxR-R4 is used and the data present: raw data (biological triplicate) for OD 600 and luminescence, normalized luminescence to OD 600 , mean and standard deviation.
The Supplementary Data 2 presents growth data and luminescence evaluation in the test tubes. The first worksheet handles OD 600 raw data (biological triplicate), processed data discounting blank, mean and standard deviation, and conversion of microplate reader (200 μL) measurement to spectrophotometer measurement (10 mm pathlength). The second worksheet handles luminescence raw data (biological triplicate), luminescence discounting blank, replace values below 0 by 0, mean and standard deviation. The third worksheet shows normalized luminescence to OD 600 , mean and standard deviation, processed data for fold change, minimum and maximum promoter activities. The fourth worksheet shows processed data for fold change.
The Supplementary Data 3 presents data on growth and GFP fluorescence for cultivations carried out in three different culture media (high sugar complex medium -PW, Luria Bertani -LB, and Mineral medium -MM), in 96-well microplate. The first, second and third worksheets handle the data in PW medium. The first, OD 600 raw data (biological triplicate), processed data discounting blank, mean and standard deviation. The second worksheet handles Fluorescence raw data (biological triplicate), normalized fluorescence to OD 600 discounting blank, replace values below 0 by 0, mean and standard deviation. The third worksheet shows processed data for fold change, fold change mean, and standard deviation. The fourth worksheet shows OD 600 in MM and LB media: raw data (biological triplicate), processed data discounting blank, mean and standard deviation. The fifth, handles fluorescence in MM and LB media: raw data (biological triplicate), normalized fluorescence to OD 600 discounting blank, mean and standard deviation, processed data for fold change, minimum and maximum promoter activities.
The Supplementary Data 4 presents data on growth and Riboflavin production in a 96-well microplate and in Erlenmeyer flasks. The first worksheet handles OD 600 and Riboflavin in microplate reader, OD 600 raw data (biological triplicate), mean and standard deviation, fluorescence, mean and standard deviation, normalized fluorescence to OD 600, mean and standard deviation. The second worksheet handles Growth data and Riboflavin evaluation in Erlenmeyer: OD 600 raw data, mean and standard deviation, fluorescence, normalized riboflavin concentration to OD 600, mean and standard deviation.

Experimental Design
To construct the autoinduction device in B. subtilis , genes luxR and luxI from Aliivibrio fischeri and the respective promoters P luxR and P luxI were cloned into the pBS3Clux plasmid upstream of the luminescence operon luxABCDE . Genes luxR and luxI were named as induction module. The plasmid was then integrated into B. subtilis genome locus sacA as single copy. Reponse promoters R1 to R7 were introduced upstream of the lux operon. R1 and R6 promoters have also been used to control GFP expression in two configurations: in cis , when both the induction module and the response module (response promoter + gfp ) were cloned into the same plasmid pBS1C and integrated at the amy locus in the genome; in trans, when the induction module was inserted at the amy locus using the plasmid pBS1C, and the response module inserted at the lacA locus in the chromosome using the plasmid pBS2E. GPF expression experiments have been performed using two different strains B. subtilis 168 and B. subtilis K07. Finally, the response promoter R6 was used to control the expression of the riboflavin operon ribDGEABHT . For that, the induction module and the rib operon under control of R6 were cloned into the pBS1C plasmid and inserted into the B. subtilis genome at the amy locus. All cloning steps have been performed using the Biobrick standard cloning.
The engineered strains were cultivated in 96-well microplates, test tubes or Erlenmeyer flasks, and periodic measurements were performed for optical density at 600 nm (OD 600 ), luminescence and fluorescence. Figure 1 summarizes the experimental design.

Plasmids and other DNA sequences
All plasmids used were derived from the Bacillus BioBrick Box [3] : pBS3Clux (BBa_K823025), pBS2E (BBa_K823027), and pBS1C (BBa_K823023). Cloning work has been performed using the standard BioBrick cloning method. luxR and luxI genes and respective promoters were PCR amplified from genomic DNA from A. fischeri. gfp was PCR amplified from pDR111_GFP(Sp) (BGSCID ECE278) [4] . Riboflavin operon ribDGEABHT was PCR amplified from genomic DNA from B. subtilis starting 250 bp upstream of the ribDG start codon, ending 40 bp downstream of the ribT stop codon including the operon transcription terminator. The 250 bp upstream of ribDG includes a mutant version of the FMN riboswitch (GGGG to AAAA at position -217) known to prevent premature transcription termination driven by the riboswitch activity [5] . Synthetic sequences corresponding to promoters in the response modules were purchased as complementary oligonucleotides. A list of plasmids and strains used is provided in Table S1 in our main paper [1] .

Bacterial strains and growth conditions
Bacterial strains used: E. coli Top10 for cloning and propagation of plasmids; B. subtilis 168, a wild type strain; and B. subtilis K07 (BGSCID 1A1133), a protease-free host.
Cultivation broths for B. subtilis strains: LB broth supplemented with 5 μg mL −1 chloramphenicol and/or 1 μg mL −1 erythromycin, when needed. A high sugar complex medium was used for GFP measurements (PW broth: 70 g L −1 sucrose, 1 g L −1 yeast extract, 25 g L −1 NaNO 3 , 0. Scale up conditions: test tubes 16 × 220 mm (d x L) were filled with 12 mL medium, and 500 mL baffled Erlenmeyer flasks filled with 50 mL LB broth were incubated at 37 °C and 220 rpm.
All experiments were performed as three biological replicates.

Growth, luminescence and fluorescence measurements
Cell growth: culture optical density at 600 nm wavelength (OD 600 ) was monitored on a 96well microplate with optically clear bottom using a microplate reader, and on a 10 mm pathlength cuvette using a spectrophotometer.
Luminescence: measurements were carried out on a microplate reader using white microplates, and an integration time of 1,0 0 0ms. GFP fluorescence: measurements were carried out on a microplate reader using black microplates, excitation at 485 nm, emission at 535 nm, and gain 100.
Riboflavin: riboflavin was quantified through luminescence on a microplate reader using black microplates. Measurements were carried out using excitation at 445 nm, and emission at 524 nm, and gain 80. A calibration curve was constructed using commercial riboflavin (Merck R7649). For riboflavin measurements in the culture medium, samples were periodically withdrawn from Erlenmeyer flasks, centrifuged at 12,0 0 0 xg for 5 min, and the flavin present in the supernatant were determined through fluorescence.
Normalization: luminescence, GFP fluorescence, and riboflavin concentration outputs were normalized to cell density by dividing each data-point by its corresponding OD 600 value.
Minimal production: defined as the lowest normalized output generated between time zero and the maximal production (minimal/initial). For identification of the minimal production, when the initial output was zero, the next sequence of outputs composed by at least four consecutive positive values was taken.
Fold change: defined as the ratio between the maximal and the minimal (initial) production of luminescence, fluorescence or riboflavin during cultivation. P veg promoter was taken as reference for promoter strength receiving the arbitrary value 1.0. The ratio between the normalized production for all other promoters to P veg was then calculated as relative promoter strength.
All raw and processed data files are available in Supplementary Data 1-4 [2] .

Python processing and plotting
Graphics shown in the Supplementary material of the related research article [1] were generated using Spyder (Python 3.7). Microsoft excel datasheet containing raw data were imported to Python for processing and plotting. Luminescence and OD 600 data were processed and plotted using the code bellow.
# Importing the libraries# import numpy as np import matplotlib.pyplot as plt import pandas as pd # Importing the dataset (

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.