Assessing the antimicrobial activity of CLEC proteins in vitro was adapted from a previously published broth dilution method (Protocol (E), [31]).

CLEC proteins were serially diluted in LB in a polypropylene 96-well plate leaving 50 μl protein dilution per well. An E. coli OP50 culture in logarithmic phase was diluted in LB to a final concentration of 100–1,000 CFUs/well, 50 μl were added to each well, and the plates were incubated at 37°C overnight. The control wells contained either only serial dilutions of the CLEC’s native buffer and OP50 (buffer control), only LB (sterility control), or OP50 in LB (growth control). Melittin (Sigma-Alrich, Cat. M2272), an antimicrobial peptide of the honeybee venom, served as positive control. The minimal inhibitory concentration (MIC) was defined as the lowest protein concentration that inhibited visibly bacterial growth, i.e. no bacterial pellet or a diffuse bacterial pellet without defined border at the bottom of the well. The wells with the resulting MICs were plated onto LB plates and incubated overnight in order to determine the CLEC’s bactericidal or bacteriostatic activity.

For testing the synergistic effect of two CLEC proteins one CLEC protein was mixed with either the second CLEC protein or with the native CLEC buffer in a polypropylene 96-well plate and serially diluted in the CLEC buffer, leaving 50 μl protein dilution per well. A Bt407 or Bt247 culture in logarithmic phase was diluted in LB to a final concentration of approx. 2,000 CFUs/well, 50 μl were added to each well, and the plates were incubated at 28°C overnight.

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