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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 371
Illegal PstI site found at 805 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 371
Illegal PstI site found at 805 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 371
Illegal PstI site found at 805 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 371
Illegal PstI site found at 805
Illegal NgoMIV site found at 690 - 1000COMPATIBLE WITH RFC[1000]
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.
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
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)
As for TFF3, the result of codon preference is shown in Fig 4.
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)
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.
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).
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.
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.
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.
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