Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Engineering AraC to make it responsive to light instead of arabinose

This article has been updated

Abstract

The l-arabinose-responsive AraC and its cognate PBAD promoter underlie one of the most often used chemically inducible prokaryotic gene expression systems in microbiology and synthetic biology. Here, we change the sensing capability of AraC from l-arabinose to blue light, making its dimerization and the resulting PBAD activation light-inducible. We engineer an entire family of blue light-inducible AraC dimers in Escherichia coli (BLADE) to control gene expression in space and time. We show that BLADE can be used with pre-existing l-arabinose-responsive plasmids and strains, enabling optogenetic experiments without the need to clone. Furthermore, we apply BLADE to control, with light, the catabolism of l-arabinose, thus externally steering bacterial growth with a simple transformation step. Our work establishes BLADE as a highly practical and effective optogenetic tool with plug-and-play functionality—features that we hope will accelerate the broader adoption of optogenetics and the realization of its vast potential in microbiology, synthetic biology and biotechnology.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Engineering and characterization of a novel light-inducible AraC.
Fig. 2: BLADE allows for the production of high-contrast bacteriographs.
Fig. 3: BLADE is compatible with pre-existing l-arabinose-responsive plasmids and strains.
Fig. 4: BLADE–sfGFP forms aggregates in the dark.
Fig. 5: Engineering an optimized and expanded family of BLADE TFs.
Fig. 6: Growth on l-arabinose can be controlled with light using BLADE TFs.

Similar content being viewed by others

Data availability

pBLADE(FP6*)–mCherry, pBLADE(FP6**)–mCherry, pBLADEONLY_A and pBLADEONLY_C have been deposited at Addgene (IDs 168048, 168049, 168050 and 168051, respectively). All other plasmids constructed in this study are also available from the corresponding authors upon reasonable request. Source data are provided with this paper.

Change history

  • 06 April 2023

    In the version of this article initially published, there was a typo in Supplementary Figure 1 where the double and single asterisks on the second and third promoters were interchanged originally; the figure has been updated in the HTML version of the article.

References

  1. Marschall, L., Sagmeister, P. & Herwig, C. Tunable recombinant protein expression in E. coli: promoter systems and genetic constraints. Appl. Microbiol. Biotechnol. 101, 501–512 (2017).

    Article  CAS  PubMed  Google Scholar 

  2. Silva, J. P. N., Lopes, S. V., Grilo, D. J. & Hensel, Z. Plasmids for independently tunable, low-noise expression of two genes. mSphere 4, e00340–19 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kong, W., Blanchard, A. E., Liao, C. & Lu, T. Engineering robust and tunable spatial structures with synthetic gene circuits. Nucleic Acids Res. 45, 1005–1014 (2017).

    Article  CAS  PubMed  Google Scholar 

  4. Benzinger, D. & Khammash, M. Pulsatile inputs achieve tunable attenuation of gene expression variability and graded multi-gene regulation. Nat. Commun. 9, 3521 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Zhao, E. M. et al. Optogenetic regulation of engineered cellular metabolism for microbial chemical production. Nature 555, 683–687 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ohlendorf, R., Vidavski, R. R., Eldar, A., Moffat, K. & Moglich, A. From dusk till dawn: one-plasmid systems for light-regulated gene expression. J. Mol. Biol. 416, 534–542 (2012).

    Article  CAS  PubMed  Google Scholar 

  7. Ramakrishnan, P. & Tabor, J. J. Repurposing synechocystis PCC6803 UirS-UirR as a UV-violet/green photoreversible transcriptional regulatory tool in E. coli. ACS Synth. Biol. 5, 733–740 (2016).

    Article  CAS  PubMed  Google Scholar 

  8. Ong, N. T. & Tabor, J. J. A miniaturized Escherichia coli green light sensor with high dynamic range. ChemBioChem 19, 1255–1258 (2018).

    Article  CAS  PubMed  Google Scholar 

  9. Jayaraman, P. et al. Blue light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic Acids Res. 44, 6994–7005 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Baumschlager, A., Aoki, S. K. & Khammash, M. Dynamic blue light-inducible T7 RNA polymerases (Opto-T7RNAPs) for precise spatiotemporal gene expression control. ACS Synth. Biol. 6, 2157–2167 (2017).

    Article  CAS  PubMed  Google Scholar 

  11. Li, X. et al. A single-component light sensor system allows highly tunable and direct activation of gene expression in bacterial cells. Nucleic Acids Res. 48, e33 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Ding, Q. et al. Light-powered Escherichia coli cell division for chemical production. Nat. Commun. 11, 2262 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Schleif, R. Regulation of the l-arabinose operon of Escherichia coli. Trends Genet. 16, 559–565 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Schleif, R. AraC protein, regulation of the l-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action. FEMS Microbiol. Rev. 34, 779–796 (2010).

    Article  CAS  PubMed  Google Scholar 

  15. Bustos, S. A. & Schleif, R. F. Functional domains of the AraC protein. Proc. Natl Acad. Sci. USA 90, 5638–5642 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Timmes, A., Rodgers, M. & Schleif, R. Biochemical and physiological properties of the DNA binding domain of AraC protein. J. Mol. Biol. 340, 731–738 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. Sheets, M. B., Wong, W. W. & Dunlop, M. J. Light-Inducible recombinases for bacterial optogenetics. ACS Synth. Biol. 9, 227–235 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wang, X., Chen, X. & Yang, Y. Spatiotemporal control of gene expression by a light-switchable transgene system. Nat. Methods 9, 266–269 (2012).

    Article  CAS  PubMed  Google Scholar 

  19. Xu, X. et al. A single-component optogenetic system allows stringent switch of gene expression in yeast cells. ACS Synth. Biol. 7, 2045–2053 (2018).

    Article  CAS  PubMed  Google Scholar 

  20. Chen, X. et al. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells. Cell Res. 26, 854–857 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Schwerdtfeger, C. & Linden, H. VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation. EMBO J. 22, 4846–4855 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Vaidya, A. T., Chen, C. H., Dunlap, J. C., Loros, J. J. & Crane, B. R. Structure of a light-activated LOV protein dimer that regulates transcription. Sci. Signal. 4, ra50 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zoltowski, B. D. & Crane, B. R. Light activation of the LOV protein vivid generates a rapidly exchanging dimer. Biochemistry 47, 7012–7019 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. de Boer, P. A., Crossley, R. E. & Rothfield, L. I. A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E. coli. Cell 56, 641–649 (1989).

    Article  PubMed  Google Scholar 

  25. Di Ventura, B. et al. Chromosome segregation by the Escherichia coli Min system. Mol. Syst. Biol. 9, 686 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Hu, Z. & Lutkenhaus, J. A conserved sequence at the C-terminus of MinD is required for binding to the membrane and targeting MinC to the septum. Mol. Microbiol. 47, 345–355 (2003).

    Article  CAS  PubMed  Google Scholar 

  27. Wachi, M. & Matsuhashi, M. Negative control of cell division by mreB, a gene that functions in determining the rod shape of Escherichia coli cells. J. Bacteriol. 171, 3123–3127 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Alyahya, S. A. et al. RodZ, a component of the bacterial core morphogenic apparatus. Proc. Natl Acad. Sci. USA 106, 1239–1244 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Pedelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Levskaya, A. et al. Synthetic biology: engineering Escherichia coli to see light. Nature 438, 441–442 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Colavin, A., Shi, H. & Huang, K. C. RodZ modulates geometric localization of the bacterial actin MreB to regulate cell shape. Nat. Commun. 9, 1280 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Bendezu, F. O., Hale, C. A., Bernhardt, T. G. & de Boer, P. A. RodZ (YfgA) is required for proper assembly of the MreB actin cytoskeleton and cell shape in E. coli. EMBO J. 28, 193–204 (2009).

    Article  CAS  PubMed  Google Scholar 

  33. Shiomi, D., Sakai, M. & Niki, H. Determination of bacterial rod shape by a novel cytoskeletal membrane protein. EMBO J. 27, 3081–3091 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Malzahn, E., Ciprianidis, S., Kaldi, K., Schafmeier, T. & Brunner, M. Photoadaptation in Neurospora by competitive interaction of activating and inhibitory LOV domains. Cell 142, 762–772 (2010).

    Article  CAS  PubMed  Google Scholar 

  35. Heintzen, C., Loros, J. J. & Dunlap, J. C. The PAS protein VIVID defines a clock-associated feedback loop that represses light input, modulates gating, and regulates clock resetting. Cell 104, 453–464 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Hunt, S. M., Elvin, M., Crosthwaite, S. K. & Heintzen, C. The PAS/LOV protein VIVID controls temperature compensation of circadian clock phase and development in Neurospora crassa. Genes Dev. 21, 1964–1974 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Mitra, D., Yang, X. & Moffat, K. Crystal structures of aureochrome1 LOV suggest new design strategies for optogenetics. Structure 20, 698–706 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Takahashi, F. et al. AUREOCHROME, a photoreceptor required for photomorphogenesis in stramenopiles. Proc. Natl Acad. Sci. USA 104, 19625–19630 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Toyooka, T., Hisatomi, O., Takahashi, F., Kataoka, H. & Terazima, M. Photoreactions of aureochrome-1. Biophys. J. 100, 2801–2809 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Grusch, M. et al. Spatio-temporally precise activation of engineered receptor tyrosine kinases by light. EMBO J. 33, 1713–1726 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1–I2 regulatory elements. Nucleic Acids Res. 25, 1203–1210 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Chen, X., Zaro, J. L. & Shen, W. C. Fusion protein linkers: property, design and functionality. Adv. Drug Deliv. Rev. 65, 1357–1369 (2013).

    Article  CAS  PubMed  Google Scholar 

  43. Reeder, T. & Schleif, R. AraC protein can activate transcription from only one position and when pointed in only one direction. J. Mol. Biol. 231, 205–218 (1993).

    Article  CAS  PubMed  Google Scholar 

  44. Aidelberg, G. et al. Hierarchy of non-glucose sugars in Escherichia coli. BMC Syst. Biol. 8, 133 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Lee, N. L., Gielow, W. O. & Wallace, R. G. Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the l-arabinose regulatory region of Escherichia coli. Proc. Natl Acad. Sci. USA 78, 752–756 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Lalwani, M. A. et al. Optogenetic control of the lac operon for bacterial chemical and protein production. Nat. Chem. Biol. 17, 71–79 (2021).

    Article  CAS  PubMed  Google Scholar 

  47. Zoltowski, B. D., Vaccaro, B. & Crane, B. R. Mechanism-based tuning of a LOV domain photoreceptor. Nat. Chem. Biol. 5, 827–834 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Bowers, L. M., Lapoint, K., Anthony, L., Pluciennik, A. & Filutowicz, M. Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340, 11–18 (2004).

    Article  CAS  PubMed  Google Scholar 

  51. Guyer, M. S., Reed, R. R., Steitz, J. A. & Low, K. B. Identification of a sex-factor-affinity site in E. coli as gamma delta. Cold Spring Harb. Symp. Quant. Biol. 45, 135–140 (1981).

    Article  CAS  PubMed  Google Scholar 

  52. Potapov, V. et al. Comprehensive profiling of four base overhang ligation fidelity by T4 DNA ligase and application to DNA assembly. ACS Synth. Biol. 7, 2665–2674 (2018).

    Article  CAS  PubMed  Google Scholar 

  53. Chung, C. T., Niemela, S. L. & Miller, R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl Acad. Sci. USA 86, 2172–2175 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Gerhardt, K. P. et al. An open-hardware platform for optogenetics and photobiology. Sci. Rep. 6, 35363 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Szeto, T. H., Rowland, S. L., Habrukowich, C. L. & King, G. F. The MinD membrane targeting sequence is a transplantable lipid-binding helix. J. Biol. Chem. 278, 40050–40056 (2003).

    Article  CAS  PubMed  Google Scholar 

  56. Doi, M. et al. Determinations of the DNA sequence of the mreB gene and of the gene products of the mre region that function in formation of the rod shape of Escherichia coli cells. J. Bacteriol. 170, 4619–4624 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. van den Ent, F., Johnson, C. M., Persons, L., de Boer, P. & Lowe, J. Bacterial actin MreB assembles in complex with cell shape protein RodZ. EMBO J. 29, 1081–1090 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  58. Kruse, T., Moller-Jensen, J., Lobner-Olesen, A. & Gerdes, K. Dysfunctional MreB inhibits chromosome segregation in Escherichia coli. EMBO J. 22, 5283–5292 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Lou, C., Stanton, B., Chen, Y. J., Munsky, B. & Voigt, C. A. Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat. Biotechnol. 30, 1137–1142 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank M. Hörner for his help with determination of the absorption spectrum of BLADE, J. Nuno de Sousa Machado for his help with size-exclusion chromatography, Y. Weber for help with characterizing the 96-well light induction plate, K.C. Huang for sharing with us the KC717 strain and S. Aoki for helpful discussions. This study was funded by the DFG (grant no. VE776/2-1 to B.D.V.), by the BMBF (grant no. 031L0079 to B.D.V.), by the Excellence Initiative of the German Federal and State Governments BIOSS (Centre for Biological Signalling Studies; EXC-294), by the European Research Council (ERC-Advanced) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 743269) and a FET‐Open research and innovation actions grant under the European Union’s Horizon 2020 research and innovation program (CyGenTiG; grant agreement no. 801041).

Author information

Authors and Affiliations

Authors

Contributions

B.D.V. and A.B. conceived the study. B.D.V. and M.K. supervised the study, and secured funding. E.R., A.B., E.B.A., N.P., M.K. and B.D.V. designed experiments and interpreted the data. E.R., A.B., M.H. and E.B.A. performed in vivo experiments. N.P. purified BLADE, and performed size-exclusion chromatography. L.E. performed the initial experiments, which validated the idea. G.S. developed the 96-well light set-up in collaboration with A.B. M.A.Ö. performed bioinformatics and computational structural biology analyses of the BLADE constructs. E.R., A.B., M.K. and B.D.V. wrote the manuscript.

Corresponding authors

Correspondence to Mustafa Khammash or Barbara Di Ventura.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 BLADE enables control of cell morphology.

a, Expected phenotypes due to overexpression of the indicated proteins. MinDΔ10 is a truncated form of the cell division MinD lacking the C-terminal membrane targeting sequence (MTS). Without the MTS, MinDΔ10 cannot associate with the membrane and remains cytoplasmic. It however maintains the ability to homodimerize26. The heterodimer formed by MinD and MinD∆10 is not able to stably bind to the membrane, because a monovalent MTS is not sufficient for this55. With MinD sequestered into the cytoplasm, endogenous MinC is no longer recruited to the membrane, and cannot counteract FtsZ, leading to the minicell phenotype. MreB is the bacterial actin homolog necessary for the establishment and maintenance of rod shape and cell wall synthesis27,56. Its assembly is regulated by RodZ, a transmembrane protein that binds MreB, altering the conformational dynamics and intrinsic curvature of MreB polymers28,32,57. It has been previously established that overexpression of MreB or RodZ leads to cell elongation and thickening27,32,58. b,d, Representative DIC images of E. coli MG1655 cells transformed with the indicated constructs grown for 4h either in the dark or under 460 nm light (5 W/m2) illumination. Images were acquired on three days with the same results. Scale bar, 5 μm. c,e,f, Quantification of cell length and width distribution for the indicated samples and conditions. Values represent mean ± SD of n = 3 independent experiments. Note that the values for the negative control (NC; pReporter_only, see Supplementary Table 1) shown in (c) are shown again in (e) to allow for the easy comparison of the length distribution of all samples. Also in (f), the NC values are the same in the upper and lower graphs. The total number of analyzed cells is: NC Dark, 1022 cells (c,e), 782 (f); NC Light, 960 cells (c,e), 674 (f); MinD∆10 Dark and Light, 880 cells; MreB Dark, 1016 cells (e, upper panel), 885 cells (f, upper panel); MreB Light, 962 cells (e, upper panel), 1006 cells (f, upper panel); RodZ Dark, 922 cells (e, lower panel), 885 cells (f, lower panel); RodZ Light, 941 cells (e, lower panel), 885 cells (f, lower panel). b-f, BLADE construct: FP4 driven by the J23101** promoter.

Source data

Extended Data Fig. 2 Comparison between AraC and BLADE.

a,b, Kinetics of mCherry expression in MG1655 cells transformed with pBAD33 (a) or pBLADE (b) and induced at t = 0 with the indicated arabinose concentrations or no arabinose (a) or with 460 nm light of the indicated intensities or left in the dark (b). c, Representative histograms showing the distribution of the mCherry fluorescence within a population of MG1655 cells transformed with either pBAD33 (orange) or pBLADE (pale blue) and induced for 4h with either 0.001% arabinose or with 0.38 W/m2 of 460 nm blue light. d, Coefficient of variation (CV) of the mCherry fluorescence levels measured by flow cytometry in MG1655 cells transformed with either pBAD33 (shades of orange) or pBLADE (shades of blue) and induced 4h with the indicated concentrations of arabinose and light intensities, respectively. e, mCherry fluorescence intensity in MG1655 cells transformed with pBLADE after repeated cycles of blue light exposure (5 W/m2) and darkness. f-h, mCherry fluorescence intensity in MG1655 cells transformed with pBAD33 after repeated cycles with the indicated arabinose concentration and without arabinose. a-b,e-f, All values were normalized to the mCherry fluorescence intensity measured in cells transformed with pReporter_only (see Supplementary Table 1; dashed line). e,f, From left to right, P = 0.46738 (e), P = 0.05474 (e), P = 0.00007 (f) and P = 4.37 × 10−9 (f). Not significant (ns), P>0.05; single asterisk (*), P < 0.05; quadruple asterisk (****), P < 0.0001. P-values P were calculated by the two-tailed, homoscedastic Student’s t test. Individual data points are the mean values of 10,000 single-cell flow cytometry events. a,b,d-f, Values represent mean ± SD of more than three biological replicates. BLADE constructs: FP6 fusion driven by the J23101* (b-d) or J23101** (e) promoter.

Source data

Extended Data Fig. 3 BLADE is dimeric under light and contacts the I2 DNA half-site.

a, Absorption spectra measured with the protein incubated 1 day (left) or 4 days (right) in the dark at 4 °C (black) or illuminated with blue light (455 nm; 50 W/m2) for 5 min at room temperature (cyan). The absorption spectrum of the blank (only medium) was subtracted from the dark and lit state spectra. b, SEC performed with purified BLADE in the dark or illuminated with 460 nm light (5 W/m2) for 30 min at 4 °C. c, Schematic representation of the synthetic promoter with the inverted I2 half-site. d, MG1655 cells transformed with pBLADE_I2 rev-mCherry grown 4h either in the dark or under 460 nm light (5 W/m2) illumination were analyzed by flow cytometry. The values were normalized to the mCherry fluorescence intensity measured in cells transformed with pReporter_only (see Supplementary Table 1; dashed line). Values represent mean ± SD of n = 3 independent experiments. The individual data points are the mean values of 10,000 single-cell flow cytometry events. Not significant (ns), P < 0.05. The P-value P was calculated by the two-tailed, homoscedastic Student’s t-test.

Source data

Extended Data Fig. 4 The foci formed by BLADE-sfGFP in the dark are aggregates and are due to the VVD moiety.

a, GFP fluorescence intensity measured in E. coli MG1655 cells transformed with a modified pBLADE in which BLADE was C-terminally fused with sfGFP grown for 4h in the dark or under 460 nm light (5 W/m2) light. The individual data points are the mean values of 10,000 single-cell flow cytometry events. b, Left, representative images of a fluorescence recovery after photobleaching (FRAP) experiment. Scale bar, 5 μm. Right, Quantification of the recovery in three independent FRAP experiments. c, Quantification of the number of cells showing aggregates for the indicated constructs and conditions. From left to right, P = 0.00451 and P = 0.09117. Not significant (ns), P < 0.05; double asterisk (**), P < 0.01. P-values P were calculated by the two-tailed, homoscedastic Student’s t-test. Values represent mean ± SD of n = 3 independent experiments. Total number of cells analyzed: n = 670 (VVD-AraCDBDWT-sfGFP), and n = 473 (VVDC108A-AraCDBD-sfGFP).

Source data

Extended Data Fig. 5 Optimal gene expression output may be obtained at intermediate TF concentrations.

a, Schematic representation of the plasmid used in step 1. Regulatory sequences (O1 and O2 half-sites) upstream of the PBAD promoter have been deleted. Right, example of a scenario for which the highest mCherry levels are obtained at intermediate cTF levels (red line). b, In step 2, the range of transcriptional output (visualized through the fluorescent reporter expression) induced by different IPTG concentrations is mapped to the rates of constitutive promoters in a library (right), allowing for the identification of a constitutive promoter that matches the desired optimal BLADE expression level (schematically shown with a red line). A plasmid bearing the IPTG-inducible promoter as well as an extended library of constitutive promoters (http://parts.igem.org/Promoters/Catalog/Anderson) was used. To minimize the potential influence of individual promoters on mRNA transcription and translation initiation, we used a ribosome binding site (RBS) containing an insulating ribozyme (RiboJ59). Plotting the mCherry fluorescence levels obtained with the constitutive promoters and with the IPTG-inducible promoter at different IPTG concentrations in the same plot, it is possible to find the constitutive promoter that best matches the expression from the IPTG-inducible one at the desired IPTG concentration. This mapping has to be performed only once for a given strain background at specific conditions, which in our case as an example was done in E. coli BW25113ΔaraC attB::lacY. Values represent mean ± SD of more than three biological replicates acquired on different days.

Source data

Extended Data Fig. 6 Setup for the characterization of BLADE TFs.

a, Light induction setup for 96-well microtiter plates containing a panel of 96 light emitting diodes (LEDs), 3D-printed holders and a metal plate for heat dissipation. The setup comprises a custom-made printed circuit board (PCB) with 96 individual LEDs of three different wavelengths (red, green and blue). Each LED can be controlled individually using a microcontroller, enabling the exposure of each well to the same light intensity, a crucial aspect for the characterization of the chimeric TFs. A milled metal plate placed in between the PCB and the LEDs dissipates the heat produced by the light induction device. A 3D-printed microplate adapter on top of the metal plate allows for the precise positioning of the 96-well plate. The light intensity of the device was calibrated using a Thorlabs PM100USB Power and Energy meter connected with a Thorlabs S170C Microscope Slide Power Sensor. b, Domain composition of the engineered light-inducible dimerization domain (VVD and VfAu1)-AraC(DBD) fusion constructs. ‘Linkers’ indicate a library of different synthetic linkers that were tested between the two domains.

Extended Data Fig. 7 IPTG-dose response curves for the VVD-AraC chimeric constructs.

IPTG-dose response curves of cells transformed with the single plasmid bearing the indicated VVD-AraC fusions and mCherry under control of PBAD deprived of all upstream regulatory elements. Cells were either kept in the dark, or illuminated with saturating blue light (465 nm; 3.85 W/m2). The lines connect the mean mCherry fluorescence values of three individual samples, which are shown in blue squares for light induced samples and black dots for the dark control. For each condition, at least n = 3 individual experiments were performed, which are shown as individual points. Only the condition for fusion VVD::G4S::AraC(DBD) incubated at 1.953 µM IPTG and with light (465 nm; 3.85 W/m2) contains two individual experiments (n = 2).

Source data

Extended Data Fig. 8 IPTG-dose response curves for the VfAu1-AraC chimeric constructs.

IPTG-dose response curves of cells transformed with the single plasmid bearing the indicated VfAu1-AraC fusions and mCherry under control of PBAD deprived of all upstream regulatory elements. Cells were either kept in the dark, or illuminated with saturating blue light (465 nm; 3.85 W/m2). The lines connect the mean mCherry fluorescence values of three individual samples, which are shown in blue squares for light induced samples and black dots for the dark control. For each condition, at least n = 3 individual experiments were performed, which are shown as individual points.

Source data

Extended Data Fig. 9 IPTG- and light-dose response of AraC(DBD)::G4S::VVD.

a, Optical density of E. coli MG1655ΔaraC cells expressing AraC(DBD)::G4S::VVD grown for 18h at 37 °C in a medium containing 2% arabinose and the indicated IPTG concentrations either in the dark or under 465 nm light (3.85 W/m2). b, Optical density of E. coli MG1655ΔaraC cells expressing AraC(DBD)::G4S::VVD grown for 17h20′ at 37 °C in a medium containing 2% arabinose and the indicated IPTG concentrations either in the dark or under 465 nm light of the indicated intensity. Bars represent mean ± SD of n = 3 biological replicates. Each data point shown in the bar plots represents a data point in a time course with measurements every 40 min, of which the average of the first three measurements was subtracted from all following time points to adjust for small differences in sample volume and inoculum. All time courses are shown in Supplementary Figs. 14 and 18.

Source data

Extended Data Fig. 10 Flow cytometry gating strategies.

a, Screenshot of the gate used for the analysis of flow cytometric data shown in Figs. 1, 3, Extended Data Figs. 14 and Supplementary Figs. 3, 5 and 6 taken from the FCSalyzer v. 0.9.1.5 α software. The SSC-A and FSC-A rectangular gate used to eliminate the debris from the cell population was set on the control strain (MG1655 cells transformed with pReporter_only) on the BD Fortessa flow cytometer and was then kept constant for all other experiments using the same cell type. For each sample, 10,000 single cells residing in the selected area were analyzed. b, Screenshot of the gate used for the analysis of flow cytometric data shown in Fig. 5, Extended Data Figs. 7 and 8 and Supplementary Figs. 11 and 12 taken from the CytExpert software. The SSC-H and FSC-H hexagon gate was drawn by eye on a control strain (E. coli MG1655 ΔaraCBAD ΔlacIZYA ΔaraE ΔaraFGH attB::lacYA177C ΔrhaSRT ΔrhaBADM Tn7::tetR kan(FRT) in the CytExpert v.2.1.0.92 and then kept constant for all experiments using the same cell type.

Supplementary information

Supplementary Information

Supplementary Figs. 1–21 and Tables 1 and 2.

Reporting Summary

Supplementary Video 1

Romano_Baumschlager_Supplementary_Video_1.

Supplementary Video 2

Romano_Baumschlager_Supplementary_Video_2.

Supplementary Data

Romano_Baumschlager_Source_Data_Supplementary_Figures.

Supplementary Dataset 1

Romano_Baumschlager_Supplementary Data Set 1.

Supplementary Dataset 2

Romano_Baumschlager_Supplementary Data Set 2.

Supplementary Dataset 3

Romano_Baumschlager_Supplementary Data Set 3.

Supplementary Dataset 4

Romano_Baumschlager_Supplementary Data Set 4.

Source data

Source Data Fig. 1

Single data points.

Source Data Fig. 3

Single data points.

Source Data Fig. 5

Single data points.

Source Data Fig. 6

Single data points.

Source Data Extended Data Fig. 1

Single-cell measurements.

Source Data Extended Data Fig. 2

Single data points.

Source Data Extended Data Fig. 3

Single data points.

Source Data Extended Data Fig. 4

Single data points.

Source Data Extended Data Fig. 5

Single data points.

Source Data Extended Data Fig. 7

Single data points.

Source Data Extended Data Fig. 8

Single data points.

Source Data Extended Data Fig. 9

Single data points.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Romano, E., Baumschlager, A., Akmeriç, E.B. et al. Engineering AraC to make it responsive to light instead of arabinose. Nat Chem Biol 17, 817–827 (2021). https://doi.org/10.1038/s41589-021-00787-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-021-00787-6

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing