Coding

Part:BBa_K3924005

Designed by: Jianfan Yang   Group: iGEM21_Tsinghua   (2021-09-30)


Defensin5


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 43
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 43
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 43
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 43
  • 1000
    COMPATIBLE WITH RFC[1000]

Profile

Name: Defensin-5(HD5)
Base Pairs: 285bp
Origin: Homo sapiens
Properties: A kind of antimicrobial and cytotoxic peptides

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

Defensin-5(HD-5) is one of the candidate therapeutic proteins we screened out to treat IBD, which is the effector element in the ternary system. Defensin-5(HD5) has antimicrobial activity against Gram-negative and Gram-positive bacteria. HD5 feeding can block the combined effect of EtOH and colitis on body weights. Colon lengths is significantly reduced by EtOH and colitis. But, HD5 can significantly attenuate the reduction of colon length.

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) [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 HD5 is acquired from Gene database (Gene ID: 1670).

Functional Verification

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)

Fig 3. Codon preference confident analysis of all candidate therapeutic proteins(Compared with GenSmart).

As for defensin-5, the result of codon preference is shown in Fig 4.

Fig 4. Codon preference confident analysis of defensin-5.

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.
[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.


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