Protocol V - Real-time monitoring of curli production in bacterial bio

This protocol describes how to use EbbaBiolight to visualise curli in biofilm forming on semi-solid agar in real-time. Curli is a functional amyloid produced by many Enterobactericeae involved in adhesion to surfaces, cell aggregation, and biofilm formation. EbbaBiolight are versatile molecules that have been reported to target various structures in the cell wall of gram-positive bacteria and the extracellular matrix of gram-negative bacteria. Curli has been identified as one of the major targets for EbbaBiolight in Salmonella biofilms using wildtype bacteria as well as curli deficient (ΔcsgA) strains. For reference, see Choong et al. We recommend using fluorescent protein tagged bacteria to be able to distinguish between bacterial cells and curli in the bacterial extracellular matrix.

Disclaimer: When adapting this technique, please make sure to include relevant controls to verify that EbbaBiolight does not affect biofilm formation, to confirm curli as EbbaBiolight binding target and to exclude pH effects. Please be aware that EbbaBiolight is suited for labelling live bacteria only and is not compatible with fixation.

Materials:

EbbaBiolight
LB broth (w/o salt)
LB agar (w/o salt)
6-well plate with cover
Curli-producing bacteria on standard culture plate

Equipment:

Incubator (28°C)
Shaking Incubator (37°C)
Microwave
Automated temperature-controlled fluorescence microscope OR Automated temperature-controlled fluorescence microwell reader

Assay Procedure:

Prepare plates with EbbaBiolight supplemented LB agar:

Microwave LB agar until it is melted
Let the microwaved agar rest at RT for 5-10 min
Add 2 µL/mL EbbaBiolight
Pour 2 mL of supplemented LB agar per well in a 6-well plate
Cover plates and let cool down at RT
Optional: Store at 4°C

Prepare bacterial inoculum:

Pick a colony from a standard culture plate
Transfer colony to LB broth
Prepare an overnight or exponential culture under continuous shaking at 37°C
Transfer 10 µl of bacterial inoculum to the centre of a dedicated well on the previously prepared 6-well plate

Real-time recording:

Incubate the plate in an automated microscope or fluorescence reader at 28°C
Program the reader or microscope to record spectra/images at defined time intervals for ca. 70 h. Use filter-sets or excitation- and emission parameters as indicated in the table below.

EbbaBiolight Fluorescence Detection:

EbbaBiolight fluorescent tracer molecules are optotracers. Unlike conventional fluorescent dyes, optotracers bind promiscuously to a range of targets with repetitive motifs. EbbaBiolight has been shown to bind to curli and cellulose in Salmonella extracellular matrix ^[1], peptidoglycan and lipoteichoic acids in the cell envelope of Staphylococci ^[2], β-glucans from S. cerevisiae and Chitin in C. albicans ^[3]. Upon binding, the fluorescence intensity of the optotracer increases. This property makes it possible to use EbbaBiolight for live fluorescent tracking of microorganisms, without the need to wash away unbound molecules. It is possible to read out fluorescence intensity at the emission maximum (Em_max) when excited at or close to the excitation maximum (Ex_max). This is useful for microscopy or fluorescence spectroscopy when straight-forward data analysis is required. Yet, due to the unique properties of the optotracers, a unique optical fingerprint is produced reflecting the specific nature of the target (sample composition) and environment (pH, osmolarity, polarity of the medium). This means that depending on the specific properties of the sample, Ex_max or Em_max can shift, or the appearance of double peaks or shoulders might indicate binding to multiple targets. We therefore recommend acquiring fluorescence excitation and emission spectra whenever possible within experimental limitations.

**Table:** EbbaBiolight spectral properties with maximum excitation (Ex_max) and emission (Em_max) when bound and recommended range for acquisition of excitation- and emission spectra as well as recommended filter sets for microscopy.
	Ex_max	Em_max	Excitation spectrum (detect at Em_max)	Emission spectrum (excite at Ex_max)	Recommended filter-sets
EbbaBiolight 480	420 nm	480 nm	300 - 450 nm	450 - 800 nm	DAPI
EbbaBiolight 520	460 nm	520 nm	300 - 490 nm	490 - 800 nm	FITC, GFP
EbbaBiolight 540	480 nm	540 nm	300 - 510 nm	510 - 800 nm	FITC, GFP, YFP
EbbaBiolight 630	520 nm	630 nm	300 - 600 nm	550 - 800 nm	PI, Cy3, TxRed, mCherry, Cy3.5
EbbaBiolight 680	630 nm	680 nm	300 - 650 nm	660 - 800 nm	PI, mCherry, Cy3.5

Access EbbaBiolight spectra

EbbaBiolight excitation- and emission spectra can be accessed here. Click or scan the QR code to access EbbaBiolight spectra:

Protocol V - Real-time monitoring of curli production in bacterial biofilms