Caulobacter crescentus andHirschia baltica naturally produce holdfast, a powerful biological adhesive. However, these organisms are notoriously difficult to work with due to their slow growth rates, making industrial-scale production impractical. Optimizing holdfast production in these native hosts is currently unfeasible, as the synthesis system and its regulation are poorly understood.

Our team embarked on a challenging journey to employ a bacteria - Escherichia coli - to produce the strongest naturally found adhesive from C. crescentus. No one has ever tried reproducing this adhesive synthesis system in E. coli, so we knew that there was a winding path of hurdles ahead of us (see Engineering). Despite all the challenges, we created a strong foundation for larger-scale manufacturing, future research, and future iGEM teams (see Results and Implementation).

Achievement no. 1: cloning the system

One of the significant necessities of our whole project idea was transferring the holdfast synthesis system from C. crescentus to E. coli. This posed a challenge since the synthesis pathway consists of 12 proteins. Nevertheless, regardless of the many setbacks we had, we developed a strategy for the transfer and cloning of this huge system (see Design and Engineering) and successfully cloned 2 parts (BBa_K5246043 and BBa_K5246046) required for transferring the holdfast synthesis system to E. coli (see Results).

Achievement no. 2: expressing the system in E. coli

The next order of business after achievement 1 was expressing the system in E. coli.

It is not a secret that E. coli, despite being one of the most beloved organisms for biomanufacturing, is not limitless. Recombinantly expressing 12 proteins simultaneously in one organism and employing it to perform a function - holdfast synthesis - is taxing on any bacteria. However, after numerous attempts (see Engineering and Results), we found expression conditions for 12 protein co-expression in the BL21(DE3). E. coli strain with gene induction with 0.5 mM IPTG followed by protein expression for 3 hours at 37°C(Fig.1.)

**Fig. 1.** SDS-PAGE analysis of CB2 system expression in BL21(DE3) at different IPTG concentrations for 3h at 37°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

After getting proteomic results (Fig.2) showing that we successfully expressed nearly all proteins except polysaccharide polymerization ones - hfsC and hfsI -, as later experiments showed, these proteins were probably substituted by paralogous proteins found in E. coli as the system without 2 parts was still producing our desired product described down below.

**Fig. 2.** Graphs depicting proteomic analysis of combined protein abundance (in absorbance units) in (a) CB2 export apparatus, (b) CB2 tetrad assembly, and (c) CB2 full systems. Expression was done in BL21(DE3) with 0.5 mM IPTG induction followed by expression for 3h at 37°C.

Achievement no. 3: E. coli producing the polysaccharide

After determining optimal expression conditions, we moved on to the biosynthesis of holdfast. We discovered that adding 1% glucose, after the expression of the holdfast synthesis proteins for 3 hours at 37°C and incubation overnight at 30°C 170 rpm, yields the formation of ring structures around the flask’s wall (Fig. 3.). We were able to successfully repeat this ring formation quite a few times (see Results).

**Fig. 3.** Appearance of rings in expression flask after the addition of 1% glucose and incubation O/N at 30°C. (a) control flasks, BL21(DE3) does not contain any target genes. No rings present. (b) flasks containing CB2 system proteins, visible rings forming.

This formed ring contained a considerable amount of N-acetyl-D-glucosamine, a characteristic component of the holdfast, whose presence we confirmed with a Wheat Germ Agglutinin dot blot assay (Fig. 4). The holdfast polysaccharide was not shed into the media but stuck onto E. coli, proving it hard to purify (see Engineering and Results)

**Fig. 4.** Dot blot assay of CB2 system expression, incubation with 1% glucose at 30°C O/N, and negative control of empty system expression. All samples contained equal amounts of cell material 1. Cells with the media 2. Supernatant from the media 3. Cell sediment resuspended in PBS. 4. PBS resuspended cells - lysed. 5. Cell lysate supernatant 6. Lysate sediment resuspended in PBS 7. Ring material resuspended in TES 8. TES resuspended ring - lysed 9. Ring lysate supernatant resuspended in TES 10. Ring lysate sediment resuspended in TES.

We were also able to confirm the previously inconclusive holdfast composition element - N-mannosamine uronic acid by knocking out the WecB gene, which is responsible for this monosaccharide production (see Engineering, Results, and BBa_K5246040). After incubation with glucose, our cultures did not form any more rings (Fig. 5).

Appearance of rings in expression flask after the addition of 1% glucose and incubation O/N at 30ºC in HMS174(DE3)ΔwecB. The empty system flask contains no target genes, CB2 system flask contains the required proteins for polysaccharide production, but in both, no thick rings are present. — **Fig. 5.** Appearance of rings in expression flask after the addition of 1% glucose and incubation O/N at 30ºC in HMS174(DE3)Δ*wecB*. The empty system flask contains no target genes, CB2 system flask contains the required proteins for polysaccharide production, but in both, no thick rings are present.

Achievement no. 4: E. coli producing biofilm-like structures

Understanding that holdfast stays attached to the producing cells led us to hypothesize that they should have noticeable morphological differences. SEM analysis revealed that the polysaccharide-producing bacteria form tight aggregates and differ morphologically from the control group. SEM pictures clearly show that holdfast-producing bacteria have altered cell wall morphology appearing bigger and more “puffy” with uneven envelope topology. (Fig. 6. 1-3., Fig. 7. 1-3.).

**Fig. 6.** SEM pictures of negative control and holdfast-producing bacteria samples. Visible biofilm-like structures. Different locations of the same CB2 sample are shown.

**Fig. 7.** Close-up of Fig.28.3. showing the “crack” in the ring material revealing tightly associated bacteria forming biofilm-like structures. Picture analyzed using Fiji software.

We saw similar results in flow cell bright-field microscopy, where it is noticeable that the CB2 sample has a considerable amount of big bacterial aggregates (Fig. 8). Biofilm-like clumps noticeable in the video can not be homogenized by pipetting or vortexing, suggesting strong associations between cells. This could explain why CB2 sample bacteria were not evenly coating the glass slide, as the aggregates prevented enough bacteria from covering the bottom of the flow cell. This can also explain why our chosen purification method was also not working (see Engineering and Results).

Fig. 8. Flow cell bright-field experiments. Noticeable aggregates of biofilm-like masses of cells can be observed floating away in the CB2 sample compared to the control.

Achievement no. 5: E. coli strain for efficient polysaccharide production

We aimed to reduce the metabolic burden on the cell membrane for polysaccharide production. We decided to deactivate one of the metabolic pathways - Enterobacterial Common Antigen (ECA) - in E. coli that produces polysaccharides, allowing substrates to be redirected towards our desired polysaccharide synthesis. We successfully created a knock-out strain WecA (see Engineering, Results, and BBa_K5246037) and tested how our holdfast production system is influenced by it.

We expressed C. crescentus CB2 holdfast system proteins, and polysaccharides in this strain and discovered that wecA gene deletion did not interfere with CB2 system protein production. Additionally, we compared it to the not-edited HMS174(DE3) strain (Fig.9.).

SDS-PAGE analysis of CB2 system expression in HMS174(DE3) and HMS174(DE3)ΔwecA at 0.5mM IPTG for 3h at 37°C and overnight at 30°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific). — **Fig. 9.** SDS-PAGE analysis of CB2 system expression in HMS174(DE3) and HMS174(DE3)Δ*wecA* at 0.5mM IPTG for 3h at 37°C and overnight at 30°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

We analyzed polysaccharide and ring production after overnight incubation with 1% glucose in an ECA-deficient HMS174(DE3)ΔwecA strain (Fig. 10). Although cells with the CB2 system grew slower than an empty control, they still produced rings with the polysaccharides, with thicker band of the ring visible in figure 9.

Appearance of rings in expression flask after the addition of 1% glucose and incubation O/N at 30ºC in HMS174(DE3)ΔwecA. The empty system flask contains no target genes, and no rings are present. A flask containing CB2 system proteins contains visible rings. — **Fig. 10.** Appearance of rings in expression flask after the addition of 1% glucose and incubation O/N at 30ºC in HMS174(DE3)Δ*wecA*. The empty system flask contains no target genes, and no rings are present. A flask containing CB2 system proteins contains visible rings.

Conclusion

We transferred the holdfast synthesis pathway from C. crescentus to E. coli, establishing a recombinant adhesive manufacturing system suitable for industrial upscale. Synhesion project is a first step towards strong, eco-friendly, and cost-effective glue production that could better the lives of millions while reducing environmental concerns from the chemical industry.

We will continue our journey towards sustainable adhesive production. We believe that the future iGEM teams (see Results and Implementation) will join us and continue developing this system or use the parts we characterized (see Parts). Let's improve not only the synthesis of the strongest natural adhesive but also the world around us.