Part:BBa_K3924029

csgA-6xHis-TFF3

This device consists of csgA as secretion peptide, Gly/Ser linker to ensure that the function of secretion peptide and therapeutic protein do not affect each other and TFF3 as therapeutic protein.

Contribution made by iGEM24_SMU-GDMU-CHINA

We have optimized the codon of the original components,you can click on the part to see details.BBa_K5378021

For the safety module,we referred to a study published in Nature Communications And the PATCH system was used for plasmid design. We first linked the gene fragments responsible for expressing curli fibers to the PBbB8k plasmid, then introduced a 6xHis-tagged linker to connect curli fibers with TFF3, and finally incorporated the TFF3 gene fragment. This configuration allows EcN to secrete and self-assemble curli fibers, linkers, and TFF3 upon reaching the intestine, forming an active domain layer on the intestinal surface. This promotes epithelial cell migration, reduces inflammatory factor levels, supports intestinal barrier repair, and alleviates hepatic encephalopathy complications.

Figure 1.our safety module design section CAPTION_HERE

Functional Verification

From the figure below, the size of each band of agarose gel electrophoresis is basically the same as the size of the target gene, indicating that the plasmid has been successfully transformed into EcN.

Figure 2.Transfer to bacterial pcr results with TFF3 plasmids

In order to confirm that curli fibers decorated with TFFs could be produced by EcN, as they can in laboratory strains of E. coli, we transformed EcN with the panel of synthetic curli plasmid constructs (Fig.3-a), in addition to a vector in place of the curli genes as a negative control. The transformed cells were cultured at 37 °C and induced with L-(+)-arabinose.

The secretion of TFF3 can be detected by Mouse trefoil factor 3(TFF3) enzyme-linked immunosorbent Assay kit. Results show that the engineered EcN was strongly induced by L-(+)-arabinose with twice as much TFF3 is produced comparing to no induction (Fig3-b).

The secretion of TFF3-fused curli was proved successful (Fig.3-c), however, In some cases, basal expression of the csgA genes was observed without induction.

A quantitative Congo Red-binding (CR) assay, normally used for curli fiber detection, indicated that CsgA-TFF3 fusions could be expressed and assembled into curli fibers under these conditions, while EcN control vector showed no CR binding(Fig3-d).

Figure 3.Functionality verification of the PEA-sensing NH3-metabolizing system.(a)Schematic representation of the process of sensing and metabolic module. EcN was co-transformed with plasmid Pcon-FeaR-Pcon-TynA and plasmid PTynA-GS via electroporation. (b)NH3 concentration after coculturing different concentration of PEA and NH4Cl with engineered EcN for 12 hours. Data shows mean±SD, n=3 independent experiments.(c)NH3 concentration after coculturing 100ng/ml PEA and 50μM NH4Cl engineered EcN for 0, 4, 8,12 and 24 hours. EcN-FeaR-TynA was transformed with only plasmid Pcon-FeaR-Pcon-TynA as the control group. Data shows mean±SD, n=3 independent experiments.

=Sequence and Features= BBa_K3924029 SequenceAndFeatures ==Usage and Biology== In order to heal the intestinal tract damage, one of notable symptoms of IBD, we adopted a special therapy expressing the therapeutic proteins controllably by E.coli Nissle 1917 (EcN) in situ. The design is based on a ternary system: sensor - secretion peptide - therapeutic proteins. [[Image: T--Tsinghua--General design of the treatment ternary system.png|center|600px|thumb|'''Fig.1 General design of the treatment ternary system''']] TFF3 is one of the candidate therapeutic proteins we screened out to treat IBD, which is the effector element in the ternary system. TFFs facilitate a significant role not only in mucosal repair but also in protecting mucous epithelia from a variety of insults in the gastrointestinal tract. The potential mechanisms to treat IBD involves anti-apoptotic properties, migration and invasion, angiogenesis, and interaction with mucins^[1]. ==Functional Verification== [[Image: T--Tsinghua--The scheme of the proof-of-concept for therapeutic proteins.png|center|600px|thumb|'''Fig 2. The scheme of the proof-of-concept for therapeutic proteins.''']] All of these proteins are worth studying, but we only chose a few proteins as a proof of concept in our actual wet lab experiments because of the time limit and the high expense of gene synthesis.
For all candidate therapeutic proteins we did codon analysis with our own software tool.(Fig 3) [[Image: T--Tsinghua--Codon preference confident analysis.png|center|600px|thumb|'''Fig 3. Codon preference confident analysis of all candidate therapeutic proteins(Compared with GenSmart).''']] As for TFF3, the result of codon preference is shown in Fig 4. [[Image: T--Tsinghua--Codon preference confident analysis of TFF3.png|center|600px|thumb|'''Fig 4. Codon preference confident analysis of TFF3.''']] The protein expression of the TFF3 was tested using SDS-PAGE and western blot. Because the TFF3 is under the tac promoter, we managed to induce the expression of EcN RGP-csgA-TFF3 using IPTG, acquired the centrifugation sediment for SDS-PAGE and performed western blot (GADPH as internal references).(Fig 5) [[Image: T--Tsinghua--Western blot for csgA-TFF3.png|center|600px|thumb|'''Fig 5. Western blot for csgA-TFF3 (GADPH as internal references).''']] After verifying that TFF3 can be successfully expressed in EcN, we also carried out preliminary animal experiments to verify its therapeutic effect. As is shown in Fig 6, the CB57BL/6 mice were induced to be IBD model by feeding DSS (3% solved in water), a widely used IBD molding agent^[2]. Then the mice are divided into 5 groups, and the normal saline (negative control group #1), EcN bacterial containing GFP gene (blank control group #2), EcN bacterial containing TFF3 gene (TFF3 non-treated experimental group #3), chitosan coated EcN (i.e. EcN@PCS, see Delivery for details) bacterial containing TFF3 gene (TFF3 treated experimental group #4) and salazosulfapyridine (positive control group #5) were applied to mice by intragastric administration on day 1, 3 and 5. The fecal occult blood representing IBD severity were measured before and after intragastric administration as a contrast. Additionally, the colon length was measured on the last day as another index of IBD severity. [[Image: T--Tsinghua--The process of animal therapeutic experiment-.png|center|600px|thumb|'''Fig 6. The process of animal therapeutic experiment. TFF3@PCS means the chitosan coated EcN, which is our delivery strategy.''']] Inferring from the weight decrease (Fig.7) and the sever fecal occult blood index (Table 2) the mice were induced to have IBD around day 0. However, the weight of all 5 groups increased after that. This indicates a recovery from IBD and a disruption of molding. Such interruption affected the following therapeutic experiment and the weight variant of control group and treatment group showed similar tendency after each time of intragastric administration (Fig.7). [[Image: T--Tsinghua--Observation of mice weight variance.png|center|600px|thumb|'''Fig 7. Observation of mice weight variance.''']] Then we compared the fecal occult blood before and after applying saline, engineered bacterial or salazosulfapyridine (Table 2). The difference between the recovery of negative control and treatment group are not significant. Only the positive control that were fed with salazosulfapyridine known to be capable to cure IBD shows a better recovery result. [[Image: T--Tsinghua--The fecal occult blood index before and after treatment-.png|center|600px|thumb|'''(Severity is classified as -, +, ++, +++ and ++++ (most severe), N/A represents a failure of feces collection)''']] As the IBD would also result in a shorter colon length, we also measured the colon length of mice to see the therapeutic effect (Fig 8). The difference between each group shows no significance due to the low amount of data and the incompletion of IBD molding, despite that the mean value of treatment group seems a little higher than negative control. [[Image: T--Tsinghua--The measurement of mice colon length.png|center|600px|thumb|'''Fig 8.The measurement of mice colon length ''']] Up to this point the in vivo experiment seems to be incapable to prove the therapeutic effect of TFF3. And we suppose that the incomplete IBD modeling causing the recovery of the mice may shade the treatment effect of the engineered bacterial. Such problem will be fixed in the future experiments with larger sample size to obtain experimental data with more translational significance. ==Reference== [1] Aamann, L., Vestergaard, E. M., & Grønbæk, H. (2014). Trefoil factors in inflammatory bowel disease. World journal of gastroenterology, 20(12), 3223–3230.
[2] Chassaing, B. , Aitken, J. D. , Malleshappa, M. , & Vijay-Kumar, M. . (2014). Dextran sulfate sodium (dss)-induced colitis in mice. Curr Protoc Immunol, 104, Unit 15.25.