Summary

We successfully characterized our modified curli-sazCA construct (BBa_25A8Q07G). The challenges we faced in detecting curli fiber may be due to issues assembling multiple csgA-sazCA fusion proteins into fibers.
We successfully created a rock by utilizing S. pasteurii’s natural biomineralization capabilities. Furthermore, we successfully sterilized this biomineralized product.

Verification of Transformants

We performed chemical transformation of E. coli K-12 with our plasmid pET_csg-sazCA_amp, which is designed to express a curli fiber and carbonic anhydrase fusion protein under IPTG induction. Additionally, the plasmid has an ampicillin resistance gene, which enables selection of transformed colonies. We streaked our recovered transformants onto LB plates and LB/ampicillin plates and incubated them overnight at 37°C.

Petri dishes showing LB/amp and LB plates with/without added DNA — Top left: LB/amp plates, K12 without added DNA
Top right: LB/amp plate, K12 with added DNA
Bottom left: LB plate, K12 without added DNA
Bottom right: LB plate, K12 with added DNA

For our bacteria without plasmid DNA, there was a smear on the LB plate and no bacteria on the LB/ampicillin plate. For our bacteria with plasmid DNA, there was a smear on the LB plate and individual colonies on the LB/ampicillin plate. This suggests that our ampicillin selection was successful and that our E. coli K-12 had likely been transformed.

We performed colony PCR and colony sequencing, both of which confirmed that our transformation was successful.

Colony PCR results — From left to right: NEB 1kb Plus DNA ladder, NEB fastload 2 ladder, transformant culture 1, transformant culture 2, transformant culture 3. The dim lines near 4 kbp suggest all three transformant cultures have plasmid.

Production of Curli Fibers

Curli fibers are extracellular proteinaceous fibers with amyloid folds, comprising key components of biofilms in Enterobacteriaceae bacteria. In E. coli, there are seven curli-specific genes (csg) organized in two operons, csgBAC and csgDEFG. The structural subunits of curli are csgA and csgB, where csgB anchors to the outer membrane and csgA is the major curli fiber monomer. The protein csgC is a periplasmic chaperone, csgE, csgF, and csgG form the secretion assembly machinery, and csgD is a transcriptional activator of the csgBAC operon. Figure 2 from Bhoite et al., 2019, shows the curli secretion pathway.

Diagram of Curli Biogenesis — Figure 2 from Bhoite et al., 2019

To test expression of our modified curli protein (BBa_259GV1IH), we used a congo red fluorescence assay inspired by Kan et al., 2019 and our conversations with the Joshi Lab. We ran the assay in quadruplicate on a 96-well plate to get better statistics.

Extract Concentration of Induced vs Uninduced Samples, with all induced vs. uninduced samples having very similar numbers except Joshi's — Graph of induced vs uninduced fluorescence readings of various cultures, with data in quadruplets. Samples 1,2 and 3 are transformant cultures. Sample 4 (joshi) is a strain we received from the Joshi Lab that expresses a different curli fusion protein and serves as a positive control. Sample 5 is untransformed E. coli K-12. Sample 6 (lb) has LB only (i.e., no E. coli) and serves as a negative control.

The increase in fluorescence between uninduced and induced cultures of our positive control indicated that our assay worked, but our transformants did not show a significant increase in fluorescence levels, indicating negligible curli fiber presence. To further investigate, we turned to computer modeling. Using AlphaFold 3, we modeled the oligomerization of our modified curli fiber and compared it against our positive control as well as to native E. coli K-12 curli. We saw significant differences in folding and assembly.

alphafold generated models — From left to right: Curli fiber monomers polymerizing normally (native E. coli K-12 curli), positive control Joshi Lab strain’s curli fiber polymerization, and our modified curli fiber polymerization. Note the pore like structure of our modified curli fibers instead of the expected linear fiber formation.

To support our hypothesis that there were protein-folding issues, we ran reverse transcription PCR to check the transcription of our gene.

gel of RT results — Gel of RT PCR results. Left to right: NEB 1kb Plus DNA ladder, K-12, Joshi, Joshi induced, curli, curli induced, curli-CA, curli-CA induced.

We created the construct pET_csg(H6)_Amp (BBa_25A80I7T), which was intended to produce unmodified curli fibers under IPTG induction. We used the congo red fluorescence assay previously described to demonstrate the presence of curli fiber, further confirming that our csgA-sazCA fusion protein faced protein folding issues.

chart of induced vs uninduced culture fluorescence — Graph of fluorescence from induced (1i, 2i) and uninduced (1, 2) cultures of our curli constructs compared to positive controls (Joshi i) and negative controls (K-12).

S. pasteurii Biomineralization

In parallel, we sought to create a biomineralized product using S. pasteurii’s natural biomineralization capabilities. S pasteurii uses the urea hydrolysis pathway, which converts urea into ammonia and bicarbonate. When Ca2+ ions are added to the solution, calcium carbonate mineralizes around the bacteria. We confirmed biomineralization under a microscope.

jagged crystal pattern under a microscope with a slight pink and green hue — *S. pasteurii* culture in CMM- under 100x magnification

frost-like crystal pattern under a microscope with a green hue — *S. pasteurii* culture in CMM- under 400x magnification

a circular shape without crystals — CMM- media, with no crystals

Material Creation and Characterization

To estimate the strength of our biomineralized material, we created molds following ASTM standards for tensile/compressive strength testing, but with scaled down dimensions. The resulting material had a cross-sectional area of 2 squared, allowing us to estimate tensile strength. However, the process of removing our biomineralized samples from the mold broke the material into several pieces, so we were unable to continue to the strength-testing stage.

ann orange block with an oar shape and a clear block of the same shape but smaller — Image of PDMS casted from a 3d printed PETG inverse. The PDMS mold is then used to create a biomineralized sample in the appropriate shape for strength testing.

Our biomineralized samples were sterilized, as outlined in our sterilization protocol, and we were able to confirm sterility via a swab test.

three containers with minerals decreasing in size — Image of our sterilized samples. Note that the right most sample, prepared in a PDMS mold, performed the best, breaking into 3 clean pieces on removal.

References

Bhoite, S., Van Gerven, N., Chapman, M. R., & Remaut, H. (2019). Curli biogenesis: bacterial amyloid assembly by the Type VIII secretion pathway. EcoSal Plus, 8(2). https://doi.org/10.1128/ecosalplus.esp-0037-2018

Kan, A., Birnbaum, D. P., Praveschotinunt, P., & Joshi, N. S. (2019). Congo Red Fluorescence for Rapid In Situ Characterization of Synthetic Curli Systems. Applied and environmental microbiology, 85(13), e00434-19. https://doi.org/10.1128/AEM.00434-19