Difference between revisions of "Part:BBa K3089008"
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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. | 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. |
Revision as of 10:58, 12 October 2023
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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 325
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 325
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 441
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 325
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 325
Illegal AgeI site found at 304 - 1000COMPATIBLE 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.
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.
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.
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.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