The adhesion system on human intestinal epithelial cells:
BBa_K4716009 to 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. 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.
Figure 3. In this figure shows that under the theoritical 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.
However, given the great diversity in the sequence of the natural cement protein of barnacle, even for cp19k only,
numerous studies around cp19k have found that the sequence of the cement protein can be more or less different even for
the same subspecies of barnacle. Therefore, this year, the cp19k we used from NCBI Nucleotide (Genbank) is different
from BBa_K3089008, but our experimental results can still provide more information about the adhesion properties of
BBa_K3089008 from the level of its interaction with human cell tissues based on the original content.
The relatively weak promoter ProA for expressing NorR in NO sensor
BBa_K4716999
After the first iteration of the design of the enzyme release module, UM-Macau decided to use Nitric Oxide, a gas
molecule that is overproduced in inflammatory environments, as an induction factor instead of the original acidic
environment induction in the adhesion module. In previous studies, including those of the previous iGEM team, a combined
NorR and PnorV system has been used as an expression initiating switch to sense the presence of nitric oxide. Based on
the design of iGEM team UZurich in 2022, and also based on the research results of Xiaoyu J. Chen et al, we have newly
added ProA, a relatively weak promoter for the NorR protein (compare with ProC and ProD), to control the expression of
NorR and invert the expression direction of this component to try to make this nitric oxide sensing system more
sensitive to nitric oxide molecules.
However, due to the late iteration of the NO sensor design, the procurement of materials for the experiments did not go
smoothly, so unfortunately we were unable to complete the experimental validation of this part before the wiki freeze.
We will continue to validate this part in the long term, and will prioritise the most important parts of the part during
the UM-Macau project conception in the next year, in order to provide future iGEMers with reliable proofs through our
team's efforts.
Figure. 4 Comparison of sensitivity to nitric oxide in different NO sensor system with ProA/ProC/ProD constitutive
promoter. The X-axis is GFP/OD600, The Y-axis is Time (h).
Contribution to part: BBa_K4716012 to BBa_K4357004
In the past year's project at UM_Macau, we have been able to improve the problem of GFP instability under acidic
conditions by replacing mCherry with GFP fluorescent protein, which is often used under acidic conditions. This year, we
found that another fluorescent protein, mSandy, has more outstanding fluorescent properties than mCherry according to
the results of S. Legault et all. In this year, we tried to replace the mCherry part of BBa_K4357004
(PSB1C3-Pasr-mCherry) with mSandy, and the combination became the new part of UM-Macau this year, BBa_K4716023
(PSB1C3-Pasr-mSandy), to explore whether it possesses better fluorescence characteristics.
However, based on the results of our experiments, our contribution to future iGEMers is not to provide them with a
better choice of fluorescent proteins, But rather, based on the lessons we have learnt, to tell future researchers who
would like to use mSandy as a label protein to be careful in choosing one based on their own laboratory conditions.
Because mSandy is a very new and niche fluorescent protein, the relevant parameters are not yet common in many lab
instruments. Therefore, when we tried to use the Olympus IX83 microscope to observe the fluorescence of under the
mCherry channal, the fluorescence emitted by mSandy was very weak, which made it difficult for us to observe the
samples.
References
- 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.
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