• Make the Fvp/PEP can degraded protein and disgest the gluten in vitroly
• Use cp19k to help bacteria to adhere to mammalian cell surface;
• Place Fvp/PEP under the control of the NO sensor, which is consist of NO promoter and NO regulator, to induce their expression under inflammation (disease condition);
• Place cp19k under the control of the Pasr promoter, which is acid induced promoter, to adapt to the human intestinal environment;

Part I, the engineering bacteria (E. coli) colonization of the mucosal layer. For this part we choose to use the cp19k and mefp-5 as the adhesive protein that present on the surface of E. coli. (by fused with CsgA). Ccp19k (moonlighting protein) from Pollicipes pollicipes participates in colonization, like double-sided glue, sticking the bacteria and the intestinal mucosal layer together to complete colonization. Mefp-5 (Mytilus edulis foot protein-5) from M-edulis is also used in the colonization to compare with the effect of cp19k. The tyrosine residues on the mefp-5 protein could be converted to amino acid residues containing the DOPA moiety by the action of tyrosinase, which is essential for the colonization of the gut by bacteria using mefp-5 expressed on the surface of E. coli.

Part II, the engineering bacteria release 33-mer-breaking enzymes, belonging to serine peptidase (S28 family). As the key pathogenicity of gluten protein is the 33-mer peptide chain, the 33-mer-breaking enzymes should be able to degrade the 33-mer peptide chain with structural properties that are difficult to be thoroughly broken down, and therefore can relieve the pathogenicity of gluten. Enzymes used in this project are endopeptidases from Flavobacterium meningosepticum, Sphingomonas capsulate, and Aspergillus niger. PEP and FVP.. Since, gluten triggers an inflammatory T-cell response in patients’ duodenum. In order to avoid T cell recognition, our groups let bacteria colonize the surface of the mucus layer to secrete breaking enzymes, and let gluten degrade before being taken up by T cells through M cells.

(1) Detection of the protein expression by the Fluorescence

Figure 1.3.1 The upper box is the design of the plasmid insert part, and the lower box is a picture of bacteria standing under a fluorescence microscope.

We can clearly see that the microscope has barely detected the mSandy's red fluorescence, and we suspect that there may be due to the following reasons:

(1) The fluorescence microscope is not optimized to detect the mSandy. We use the mCherry channel instead of detecting the fluorescence of mSandy,. aAlthough the wavelengths of light differ by only a few nanometers, this may reduce the sensitivity during the detection.:
mSandy 2 (𝜆em = 606 nm, quantum yield = 0.35)
mCherry (𝜆em = 609 nm, quantum yield = 0.23)

(2) We designed that the location of mSandy is between promotor and cp19k proteins, and may affect the conformation of mSandy proteins and affect the signal. However, if mSandy is expressed after cp19k, there is a high probability of affecting the adhesion effect of cp19k. Hence, o mSandy's location became a problem remained to be solved. As an alternative, we can swap the reporter protein for a small tag (such as 6xHis) and the protein expression can be detected by Western blotting.

(3) After seqeuencing, we found a mutation on the J23119 promoter, this may affect he transcription of the target gene. However, even the protein expressed by the Pasr promoter has not detected fluorescence.

Figure 1.3.2 The upper 2 boxes areis the design of the plasmid insert part, and the lower box is a picture of bacteria standing under a fluorescence microscope.

There are only 1 photo each of (1) pET-J23119-Fvp-his tag (2) pET-J23119-PEP-his tag bacteria, because of the relationship between technology and time (the part of the NO sensor is the part added during the second design), the plasmids related to the NO sensor have not been built for the time being, and there are no fluorescent photos of the related bacteria.

We found that only bacteria containing Fvp plasmids had partial green fluorescence, while almost no fluorescence compared to PEP-expressing bacteria, and we hypothesized that perhaps E. coli was more receptive to the expression of the Fvp protein (the size of the Fvp protein is slightly smaller than PEP). However, we can see that the fluorescence distribution is not uniform, and no fluorescence is found outside the bacterial cell, only there are obvious fluorescent spots in the bacterial cell, which may indicate that the protein is not successfully secreted outside the cell when the photo is taken.

(2) The adhesion assay to determine the function of the Cp19k aAdhesion protein

Brief introduction: We put the same bacterial concentration (OD600 = 0.5 ± 0.02) on the surface of different materials to culture for 1h and 2h, and then wash off the floating and non-unadherent bacteria with 1XPBS, take pictures and count the number of bacteria left on (sticking) the surface of the material.

Experiment I Adhesion assay with pH = 7 culture condition (1h)
Experiment II Adhesion assay with pH = 5 culture condition (1h)
Experiment III Adhesion assay with Caco-2 intestine epithelial cells (2h)

Summary of the adhesion protein result:

• Under acidic conditions, the Pasr promoter expressed the Cp19k and increase the adhesion ability of the bacteria.
• The adhesion effect of cp19k enhanced by collagen coating.

Experiment I Adhesion assay with pH = 7 culture condition (Compare the adhesion properties on different plasmids )

Figure 1.3.3 The figure shows the count of bacteria under a microscope (100X objective and 10X eyepiece, 1344pixel × 1024pixel). After a period of culture, we washed the surface of bacteria where bacteria lived once with 1XPBS.

Figure 1.3.4 The figure shows the count of bacteria under a microscope (100X objective and 10X eyepiece, 1344pixel × 1024pixel). After a period of culture, we washed the surface of bacteria where bacteria lived once with 1XPBS.

Graph 1.3.2 G = Glass, G + C = Glass + Collagen.

Experiment III Adhesion assay with Caco-2 intestine epithelial cells

Figure 1.3.5 The figure shows the count of bacteria under a microscope (100X objective and 10X eyepiece, 1344pixel × 1024pixel). After a period of culture, we washed the surface of bacteria where bacteria lived once with 1XPBS.

Graph 1.3.3 G = Glass, G + C = Glass + Collagen.

We observed that:

• There is no change of the adhesion after the expression of cp19k. This may be related the original affinity of the bacteria on the cells is strong, or due to the complex extracellular matrix environment of the mammalian cells.

(3) Effect of pH on bacteria growth curves with different plasmid

Brief introduction: We incubated the same number of bacteria overnight in LB medium (with chloromycetin as antibiotic) with a certain pH gradient (pH=4.75-6.5, interval 0.25) for 20 h, and finally the OD600 value of the bacterial solution was measured by microplate reader.

Summary of the result:

• Bacteria with J23119 promoter expression plasmids survive better
• Within each plasmids, there is no strong inhibition of cell growth at different pH condition, except Psar-cp19k and Psar-control, this indicate the acid promoter may affect the bacteria growth under strong acid condition (pH <5)

Graph 1.3.4 All the graphs here show the growth of our engineering bacteria. (A) Summary of growth. (B) J23119 promoter (Part: BBa_J23119) bacterial growth expressing cp19k. (C) Bacterial growth containing J23119 promoter but not expressing special proteins. (D) Containing Pasr promoter (Part: BBa_K1231000) bacterial growth without expressing specific proteins. (E) Growth of bacteria expressing cp19k by Pasr promoter.

(4) Gibson assembly results

From the synthesis of plasmids at the beginning, to the addition of new fragments (such as the addition of NO sensor), and the modification of some plasmids, it is necessary to go through PCR and Gibson assembly experiments. We have both successful and failed plasmid builds.

Figure 1.3.6 One result of template DNA running gel. After completing the PCR procedure for the template DNA, based on the sequence size, we determined if the DNA was the correct size for the next step for Gibson assembly.

Figure 1.3.7 Photographs of some completed Gibson's plasmids and transfected into medium for E. coli. We can see some without long colonies, such as the left image; There are some that have successfully grown colonies, as shown in the figure on the right.

For the analysis of the experiment:

• Perhaps it is a procedural problem (see Protocol for details), such as the possibility that the temperature and time may not be suitable for the ligase we are using;
• Maybe it's a problem with our personnel operation, most of the members are not skilled in experimental operation, maybe the experiment fails because of ignoring some small details during the experiment, such as mix is not mixed;
• Or maybe the template DNA is not pure enough, causing Gibson to fail; Wait a minute.
• But the experiment continues...