This document describes several experimental procedures used to study curli production and biofilm formation in bacteria: 1) Congo Red assays and western blot analysis are used to assess curli production in bacterial strains. 2) Purification methods are described to isolate curli subunits from bacterial cells, either under native or denaturing conditions. 3) Polymerization assays use purified curli subunits and fluorescent dyes to study fiber formation and seeding processes in vitro. 4) Biofilm formation assays examine the role of curli in supporting ring-shaped and pellicle biofilms at the air-liquid interface.

2. Congo Red (CR) Assays to
Assess Curli production
Western Blot Analysis
of Curli
Purification of Curli
subunits
Polymerization Assay
Curli dependent Biofilm
Formation
CR assay for bacteria on YESCA CR agar
Post-staining CR assay
Western Blot Analysis of Bacterial Lysates
Plug Western Blot Analysis
Non-denaturing Purification
Denaturing Purification
Polymerization from monomers
Seeding assay
Microtiter Plate assay for ring-shaped Biofilm
Pellicle Biofilm Assay
EXPERIMENTAL PROCESS

3. Congo Red (CR) Assays to Assess Curli production
* CR assay on YESCA CR Agar
* Post-staining CR assay
• Wild-type curli-producing
stain red.
• Curli defective mutants
are usually pink or white.
• E. coli mutants with hyper
curli production
sometimes stain dark red.

4. Western Blot Analysis of Curli
Whole cell
plugs
• Antibodies (raised in rabbit):
 CsgA antibody (Proteintech) against
purified CsgA
 CsgB antibody (Proteintech) against
peptide fragment of second repeating
unit of CsgB
• Anti-rabbit secondary antibody: peroxidase
antibody producted in goat

5. Western Blot Analysis of Curli

6. Purification of Curli subunits
Method Non-denaturing Denaturing
• Advantage
o Avoid denaturation of target
protein
o Less time-consuming
o Cell can be stored in a
long term
• Disadvantage
o Time-consuming
o Supertanant cannot be
stored > 1 day
o Low yield
• Stimulation

7. Purification of Curli subunits
* Non-denaturing Purification of CsgA or truncated CsgB
• Expression strain: LSR12/pMC1/pMC3
• This strain resistant to Ampicilin (Amp) and Chloramphenicol (Cm)
• CsgA (truncated CsgB) can be collected by Affinity Chromatography
LSR12
CsgDEFG csgBA
deletion mutation
in E. coli C600
pMC1
cloning csgG into the NcoI and BamHI
sites of pTrc99A plasmid
pMC3
cloning full-length C-terminal His6-
tagged CsgA into the NdeI and EcoRI
sites of pHL3 plasmid.
• Protein experession is induced with IPTG

8. Purification of Curli subunits
* Denaturing Purification of CsgA or CsgB (bacterial lysate)
• Expression strain: NEB C2566/pNH11 (CsgA)
NEB C2566/pAN1 (CsgB)
• This strain resistant to Ampicilin (Amp) and Chloramphenicol (Cm)
• Use GdnHCl to lyse the cell
• Collect protein by Affinity Chromatography
NEB C2566: T7 expression strain of E. coli
pAN1
plasmid made by cloning C-
terminal His6-tagged CsgB
without Sec signal sequence
into pET11d vector
pNH11
plasmid made by
cloning C-terminal
His6-tagged CsgA
without Sec signal
sequence into pET11d
vector

9. Polymerization Assay
samples
* Polymerization from monomersControl
- Control: load a well with only buffer
- Samples: load 100µl of purified protein
with various concentration of the same
protein
+ 2µl of ThT solution
Fluorescent plate reader
λexcitation ~ 438nm
λemission ~ 495nm

10. Polymerization Assay
* Polymerization from seeding
• Prepare seeds
1-3 days old, fresh
CsgA (CsgB) fibers
Sonicate on ice
15s x 3 times
seeds
• Seeding assays
100μl monomeric protein
100μl Kpi buffer
samples
control
+ 2-10% seeds
+ 2μl ThT solution

11. Polymerization Assay
𝒕 𝟏/𝟐
𝒏𝒐 𝒔𝒆𝒆𝒅𝒊𝒏𝒈
> 𝒕 𝟏/𝟐
𝒔𝒆𝒆𝒅𝒊𝒏𝒈

12. Curli dependent Biofilm Formation
• E. coli MC4100 forms a ring-shaped biofilm at the air-liquid interface on the inner
surface of glass tube (microtiter plate wells)
• Some E.coli and Salmonella spp also form a pellicle-like biofilm floating on the
air-liquid interface
• Curli defective mutants are unable to form biofilm
=> Biofilm formation is depend on curli