Difference between revisions of "Part:BBa K4716009"

Latest revision as of 11:09, 12 October 2023

pSB1C3-Pasr-CsgA-cp19k-mSandy

On the direction for our gene expression, there is an Pasr promoter (BBa_K4716000) which is the pH-responsive promoter native to E.coli, inducing transcription in human duodenum region’s relatively acidic environment (pH 5~6), RBS (BBa_B0034) is the ribosome binding site, the target gene - CsaA and mSandy2, Cp19k are connected with the sequence of GS linker, and the mSandy2 is the reporter gene; The double terminator (BBa_B0015) is the terminater we use in this circuit, BioBrick prefix and BioBrick suffix are presented at the beginning and end of the whole insert gene, respectively. In the opposite direction, the cat promoter controls the CmR gene which is the selective marker, carrying chloramphenicaol resistance gene for our screening object. Followed by Lambda T0 terminator, there is also a replication origin for the whole plasmid.

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] 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.doi:10.1007/s10565-005-0085-6

Sequence and Features