A Biofilm Free E. coli Chassis for Use in

Continuous Culture

Chris N. Takahashi Department of Electrical Engineering University of Washington

Seattle, WA 98195

Email: cnt@u.washington.edu

Eric Klavins

Department of Electrical Engineering University of Washington Seattle, WA 98195

Email: klavins@u.washington.edu

a)

WT BF019 c)

b)

WT BF019

selective pressures in directed evolutions. In continuous culture, substrate concentrations and metabolic byprod- ucts reach a steady state eliminating the drawbacks of batch culture. However, continuous culture selects strongly for phenotypes that are resistant to washout, such as flocculent yeast or adherent biofilms in E. coli [2]. Lab strains of yeast that are resistant to flocculation [3] are readily available, however a strain of E. coli that does not form biofilms in continuous culture has not yet been reported. Here, we have developed a five gene/operon E. coli knockout strain BF019 that is resistant to biofilm formation in continuous culture. We show that wild type E. coli can form disruptive biofilms in as little as 48 hours, while our novel strain remains biofilm free for at least two weeks, after which we ended the experiment. Strain BF019 and our previously reported [4] open-source turbidostat design [5] form a platform enabling characterization of long-term dynamics as well as directed evolution of better

WT BF019

Fig. 1. Comparison of wild type and BF019 a) Cross-sectional schematic of a continuous culture vessel. Wild-type E. coli have formed a biofilm causing the washout of planktonic cells. BF019 cannot form biofilms and therefore stays in a planktonic state. b) Wild type vs. BF019 biofilm formation on glass culture tubes used in continuous culture. c) A standard crystal violet stained plate assay [1] showing WT vs BF019. Wild type E. coli readily forms biofilms near the gas-liquid interface and on the bottom of the well (not shown), while BF019 does not form detectable biofilms.

Abstractâ??Continuous culture provides constant culture conditions enabling better characterization of synthetic circuits as well as constant selective pressures for directed evolution. When characterizing synthetic circuits over generational timescales (tens of minutes to hours), cellular metabolism impacts batch culture conditions, which can act as a disturbance to synthetic circuits and change

substrate utilizers and more tolerant strains required for consolidated bioprocessing.

REFERENCES

[1] J. H. Merritt, D. E. Kadouri, and G. A. Oâ??Toole, â??Growing and Analyzing Static Biofilms,â? in Current Protocols in Microbiology (R. Coico, T. Kowalik, J. Quarles, B. Stevenson, and R. Taylor, eds.), Hoboken, NJ, USA: John Wiley & Sons, Inc., Aug. 2011.

[2] J. D. Bryers, â??Biofilm formation and chemostat dynamics: Pure and mixed culture considerations,â? Biotechnology and Bioengi- neering, vol. 26, pp. 948â??958, Aug. 1984.

[3] W. S. Lo and A. M. Dranginis, â??FLO11, a yeast gene related to the STA genes, encodes a novel cell surface flocculin,â? Journal of Bacteriology, vol. 178, pp. 7144â??7151, Dec. 1996.

[4] C. N. Takahashi, A. W. Miller, F. Ekness, M. J. Dunham, and E. Klavins, â??A low cost, customizable turbidostat for use in synthetic circuit characterization,â? ACS Synthetic Biology, vol. 4, no. 1, pp. 32â??38, 2015.

[5] â??Klavins lab hardware.â? http://klavinslab.org/hardware.