Part:BBa_K3089008

rBalcp19K

One of our adhesives. Barnacle cement proteins are very promising in making biomedical bioglues. rBalcp19K had the properties of both self-assembly and adhesion.

This composite part is meant to express rBalcp19k gene under T7 promoter. This part is also expressed in Pichia pastoris (BBa_K3089016) after codon optimization. rBalcp19k, the barnacle adhesive protein from Balanus albicostatus, its homologous gene serves as adhesive proteins, also playing the role of interfacial adhesion in a way that mfp5 does in mussels(Liang, et al. 2015) (Figure 1). We linked them together to achieve better adhesive ability, because rBalcp19k can self-assemble into aggregated nanofibers at acidic pHs. This composite part would be a promising new generation of bio-inspired adhesives for a wide range of applications.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 325
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 325
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 441
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 325
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 325
  Illegal AgeI site found at 304
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Three different experiments were done to characterise the BBa_K3089008 biobrick:

• protein expression
• protein purification
• Surface coating analysis

Protein expression

rBalcp19k was cloned into pET28b and expressed in E.coli BL21(DE3) Rosetta by 500μM IPTG for 5h at 37℃. In order to detect its expression, whole cells were collected after induction by centrifuging and prepared for SDS-PAGE. Results showed that obvious protein band of rBalcp19k(~19KDA) could be observed on lane WC compared with lane pET28b (pET28b empty vector), which means the expression of this protein is well in BL21(DE3) Rosetta.

Figure 2. Detection of the expression level of all recombinant proteins by SDS-PAGE.(A) SDS-PAGE of whole-cell lysates of all recombinant proteins. Red arrows show the predicted place of certain proteins. (B) Protein SDS-PAGE bands optical densities were measured by quantitative densitometry of SDS-PAGE of whole-cell aliquots.

Protein purification

We expressed it in E.coli BL21(DE3) Rosetta, and we found bands of rBalcp19K appeared between 15kDa and 25kDa on 12% SDS-PAGE gel (Figure 5A), which meant it was successfully expressed and purified under native condition. In SDS-PAGE of rBalcp19k, we found various unexpected bands, even if we used higher concentration imidazole as washing buffer, we still couldn’t get rid of them. It seemed like they were polymers of rBalcp19k caused by self-assembly. The final yield was 2mg/L.

Figure 3A. SDS-PAGE of purified rBalcp19k (A) by affinity chromatography under native conditions. Lanes: M, protein molecular weight marker; NC, whole-cell sample of pET28b empty vector; WC, whole-cell sample of recombinant protein rBalcp19K; S, soluble cell fraction; W1, fraction.

Surface coating analysis

After obtaining a small number of recombinant proteins, surface coating analysis (see methods), the qualitative assessment of the surface adsorption ability of recombinant proteins, was performed on 2 of the most commonly used bio-related surfaces: hydrophilic glass slides and hydrophobic polystyrene tissue culture plates. As shown in Figure 3, rBalcp19K exhibited lower surface absorption abilities than rBalcp19k-mfp5, whereas almost all absorbed BSAs were washed away. Basing on our current understandings of Mfp5, we were able to predict the adhesiveness of this protein, especially when it is combined with different proteins, making it more adaptable in many environments.

Figure 4. Surface coating analysis of recombinant proteins on hydrophilic glass slides (left) and hydrophobic polystyrene (PS) plates (right).

Reference

Liang, C., et al.(2015). Protein Aggregation Formed by Recombinant cp19k Homologue of Balanus albicostatus Combined with an 18 kDa N-Terminus Encoded by pET-32a(+) Plasmid Having Adhesion Strength Comparable to Several Commercial Glues. PLoS One 10(8):e0136493.

Team UM-Macau 2023: Contribution of BBa K3089008 This year, we have introduce a new part into the iGEM: The relatively acidic environment induced adhesion system (BBa_K4716009) which is drived by the pH sensitive promoter Pasr promoter (BBa_K1231000), and use the adhesion protein cp19k (BBa_K3089008) from barnacle, an edible marine species. By virtue of the acidic pH of the duodenal segment of the small intestine due to its proximity to the lower end of the stomach, we chose the Pasr promoter as the initiator switch, and by CsgA to present the surface display technology of intestinal probiotics, to express the adhesion protein cp19k on the surface of the E.coli DH5-alph used in our experiments, so as to make it stronger in adhesion properties compared to the wild type.

Figure 1. The circuit design of pSB1C3-Pasr-cp19k-mSandy.

In most of teh previous study, the genetic engineering practices related to cp19k have generally used it as a purely biological adhesive material acting on inorganic substances rather than the adhesion of organisms. In contrast, UM-Macau has this year for the first time studied the interaction between this adhesion system and organisms based on the induction of proximate environmental conditions in the human body. Therefore, in addition to the adhesion experiments between engineered bacteria and inorganic materials (glass slides), which were introduced as a new part of the system. We also performed adhesion experiments between engineered bacteria and slides covered with collagen, a basal exposure protein that mimics the partial loss of small intestinal epithelial cells in celiac disease patients, and slides covered with the colorectal cancer cell line Caco-2 under the pH values of 5 and 7. In the process of gradually providing experimental conditions close to the real small intestinal environment, we have also gradually verified the feasibility and validity of combining the system with organisms and interpreted the data with reasonable information in conjunction with the growth of the engineered bacteria and other parameters, which will become some useful data and information for the iGEMer in the future to refer.

Figure 2:This figure shows that under the theoretical suitable pH value of Pasr promoter, the difference of adhesion effect between glass and glass+collagen. After incubating our engineered E.coli DH5-alpha with BBa_K4716100 for 1 hour then use the slow-moving water, the observation shows the cp19k can have better adhesion effect when the glass slides are coated with collagen.

Figure 3: In our modeling work, the result of using Discover Studio also proves the adhesion protein cp19k can interact perfectly with collagen VII in the structure of mucous layer’s ECM. This figure shows the best pose of the interaction between collagen VII and cp19k.

References:
[1] Liang, C., et al.(2015). Protein Aggregation Formed by Recombinant cp19k Homologue of Balanus albicostatus Combined with an 18 kDa N-Terminus Encoded by pET- 32a(+) Plasmid Having Adhesion Strength Comparable to Several Commercial Glues. PLoS One 10(8):e0136493.
[2] Dressman, J. B., et al. (1990). Pharmaceutical Research, 07(7), 756–761.doi:10.1023/a:1015827908309
[3] Vita, N,  Borne, R,  Fierobe, H-P.  Cell-surface exposure of a hybrid 3-cohesin scaffoldin allowing the functionalization of Escherichia coli envelope. Biotechnology and Bioengineering.  2020; 117: 626–636. https://doi.org/10.1002/bit.27242
[4] Sambuy, Y., De Angelis, I., Ranaldi, G. et al (2005). The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol Toxicol 21, 1–26. https://doi.org/10.1007/s10565-005-0085-6