Difference between revisions of "Part:BBa K3924000"

Revision as of 06:52, 21 October 2021

TFF1

aaa

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
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COMPATIBLE WITH RFC[21]
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COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 13

Profile

Name: TFF1
Base Pairs: 369bp
Origin: Homo sapiens
Properties: One of human cytokine from The trefoil factor (TFF) family secreted by mucus-producing cells.

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.

Fig.1 General design of the treatment ternary system

TFF1 is one of the candidate therapeutic proteins we screened out to treat IBD, which is the effector element in the ternary system. TFF 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 involving anti-apoptotic properties, migration and invasion, angiogenesis, and interaction with mucins.

Design and Construction

According to literature research we chose 10 candidate proteins for IBD treatment. Table 1. List of candidate therapeutic proteins

Part Name	Element Name	Reference
BBa_K3924000 BBa_K3924001 BBa_K3924002	TFF1/2/3	[1][2]
BBa_K3924003	Chromofungin(CHR: CHGA47-66)	[3]
BBa_K3924004	IL10	[4]
BBa_K3924005	Defensin-5(HD5)	[5]
BBa_K3924006	GLP2	[6]
BBa_K3924007	Fgl2	[7]
BBa_K3924008	SOCS1-KIR	[3]
BBa_K3924009	tkip	[3]
BBa_K3924037	G-CSF	[8][9]
BBa_K3924038	GM-CSF	[8][10]
BBa_K3924039	Beta-defensin 4A(hBD2)	[11]

The sequence of TFF1 is acquired from Gene database (Gene ID: 7031). The sequences were synthesized on pMV plasmids by biological companies and cloned into pEGFP & RGP-TMAR plasmids by ourselves for the following experiments. PCR technique and HiFi assembly method were frequently utilized. Eventually, we have successfully constructed the plasmids RGP-csgA-TFF1 as the best carrier of our treatment protein. The plasmids were amplified inside E. coli DH5α and imported into E. coli Nissle through electrotransformation. The protein expression of the TFF1 was tested using SDS-PAGE and western blot.

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 TFF1 we did codon analysis with our own software tool, and the results are shown in Fig.

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] Praveschotinunt, P., Duraj-Thatte, A.M., Gelfat, I. et al. Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut. Nat Commun 10, 5580 (2019).
[3] La Manna, S., Di Natale, C., Florio, D., & Marasco, D. (2018). Peptides as Therapeutic Agents for Inflammatory-Related Diseases. International journal of molecular sciences, 19(9), 2714.
[4] Li, M. C., & He, S. H. (2004). IL-10 and its related cytokines for treatment of inflammatory bowel disease. World journal of gastroenterology, 10(5), 620–625.
[5] Shukla, P.K., Meena, A.S., Rao, V. et al. Human Defensin-5 Blocks Ethanol and Colitis-Induced Dysbiosis, Tight Junction Disruption and Inflammation in Mouse Intestine. Sci Rep 8, 16241 (2018).
[6] Duan, L., Rao, X., Braunstein, Z., Toomey, A. C., & Zhong, J. (2017). Role of Incretin Axis in Inflammatory Bowel Disease. Frontiers in immunology, 8, 1734.
[7] Zhu, Y. , Jie, Z. , Yi, F. , Chen, L. , Zhang, L. , & Fei, Y. , et al. (2018). Control of intestinal inflammation, colitis-associated tumorigenesis, and macrophage polarization by fibrinogen-like protein 2. Frontiers in Immunology, 9, 87-.
[8] Guidi, L., Mocci, G., Marzo, M., & Rutella, S. (2008). Treatment of Crohn's disease with colony-stimulating factors: An overview. Therapeutics and clinical risk management, 4(5), 927–934. https://doi.org/10.2147/tcrm.s2756.
[9] Vanz, A. L. , Renard, G. , Palma, M. S. , Chies, J. M. , Dalmora, S. L. , & Basso, L. A. , et al. (2008). Human granulocyte colony stimulating factor (hg-csf): cloning, overexpression, purification and characterization. Microbial Cell Factories, 7(1), 1-12.
[10] Malekian, R., Jahanian-Najafabadi, A., Moazen, F., Ghavimi, R., Mohammadi, E., & Akbari, V. (2019). High-yield Production of Granulocyte-macrophage Colony-stimulating Factor in E. coli BL21 (DE3) By an Auto-induction Strategy. Iranian journal of pharmaceutical research : IJPR, 18(1), 469–478.
[11] Koeninger, L. , Armbruster, N. S. , Brinch, K. S. , Kjaerulf, S. , & Wehkamp, J. . (2020). Human β-defensin 2 mediated immune modulation as treatment for experimental colitis. Frontiers in Immunology, 11, 93.

@@ Line 18: / Line 18: @@
 ==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: General design of the treatment ternary system.png|'''Fig.1 General design of the treatment ternary system''']]
+[[Image: General design of the treatment ternary system.png|center|600px|thumb|'''Fig.1 General design of the treatment ternary system''']]
 TFF1 is one of the candidate therapeutic proteins we screened out to treat IBD, which is the effector element in the ternary system. TFF 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 involving anti-apoptotic properties, migration and invasion, angiogenesis, and interaction with mucins.
 ==Design and Construction==