Composite

Part:BBa_K4829013

Designed by: Aditya Kamath Ammembal   Group: iGEM23_IISc-Bengaluru   (2023-10-05)


IVT of this sequence produces mRNA coding for a dAb against IL8

This composite biobrick is one of multiple combinations possible using our modular mRNA-based protein expression systems. We have not produced this using IVT. However, considering our success with BBa_K4829003, we can confidently say that this will produce the required dAb. We would like to emphasise further, that though this composite biobrick has used the CD33 signal peptide, we urge the users of the dAb for this application(mRNA protein production) to try out various different signal peptides!

Usage and Biology

  • This part is designed for In Vitro Translation/Transcription. If this DNA fragment in a backbone is transfected into a mammalian cell, it WILL NOT produce a protein.
  • We recommend that anyone using this construct use modified nucleotides: N1-Methylpseudouridine, and cap the mRNA with CleanCapAG, to improve translational efficiency and reduce immune response to the mRNA.
  • It has a T7 promoter (BBa K4829000), a 5'UTR(BBa K4829004), a coding sequence (which has a signal peptide(BBa K4829001) along with the dAb(BBa K4829007)), a 3'UTR(BBa K4829005), and a polyA tail(BBa K4829006).
  • On In Vitro Transcription, followed by purification, the mRNA produced can be used to transfect HeLa cells with Lipofectamine messengerMax. After 6 hours, the protein will be detected in the supernatant.
  • This part is intended to be used as an mRNA therapeutic for endometriosis (for further information, you may look at our wiki). This part is also intended for conditions like triple-negative breast cancer. (Check the references).
  • We recommend that anyone suing this construct use modified nucleotides: N1-Methylpseudouridine, and cap the mRNA with CleanCapAG, to improve translational efficiency and reduce immune response to the mRNA.

This composite biobrick is meant to be used in In Vitro Transcription/Translation. We would like to notify all users of this part that this DNA WILL NOT produce protein on transfection into mammalian cells. This specific dAb is expected to be a neutralising one. We have not used it for neutralising purposes ourselves yet. However, the potential use of this dAb would be to block IL8 either in disease processes or possibly even in cell biology assays/related studies. Here are some potential uses of a dAb that binds to IL-8:

  • Therapeutic Applications:

Anti-inflammatory treatments: Given that IL-8 is involved in promoting inflammation, a dAb that neutralizes IL-8 could be used as a therapeutic agent to treat inflammatory conditions like chronic obstructive pulmonary disease (COPD), asthma, or rheumatoid arthritis.

  • Cancer treatments: IL-8 can promote tumor growth, angiogenesis, and metastasis. Neutralizing IL-8 with a dAb might inhibit these processes and could be used as an adjunct therapy in cancer treatment.
  • Treatment of infectious diseases: IL-8 plays a role in the pathogenesis of certain infections. Neutralizing IL-8 might modulate the host response, potentially reducing tissue damage and improving the outcome.
  • Diagnostic Applications:
  • Biosensors: dAb’s that bind IL-8 could be used in biosensors to detect and quantify IL-8 in biological samples, providing insights into inflammation or other pathological processes.
  • Imaging: Labeled dAb’s could be used in imaging modalities, like PET or MRI, to visualize sites of inflammation or tumors where IL-8 is overexpressed.
  • Research Tools:
    • Protein interaction studies: The dAb could be used to study the binding characteristics of IL-8 with its receptors or other interacting proteins.
    • Structural biology: Because of their small size and ability to bind specific epitopes, dAb’s can be excellent tools for crystallizing proteins or protein complexes for structural studies.
    • Cell-based assays: dAb’s could be used in cellular assays to study IL-8 signaling pathways or to neutralize IL-8 activity.
  • Drug Screening:

dAb’s can serve as a tool for high-throughput screening assays to identify compounds that interfere with IL-8 binding or function.

  • Drug Delivery:

Due to their small size and specific binding properties, dAb’s can be conjugated to drugs or other therapeutic agents and used to target them to specific sites where IL-8 is present.

  • Vaccine Development:

Although more exploratory, dAb’s could be used as a scaffold for vaccine development, especially if they can induce a neutralizing response against IL-8 or associated pathogens. We have not included his tag/FLAG tag in this sequence. However, it would be prudent to do so, for purification purposes. The specific features of this dAb have been modelled by our dry lab team, and the details may be found on our dry lab page. A brief summary of the details is mentioned below.

The image shown here is of the model of the dAb, predicted using Alphafold

  • il8nbdock.png 
 Figure 1. IL8(red) binding to our dAb(green)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 191
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 286

Functional Parameters

The functional parameters we have been able to measure are the Gibbs free energy of binding and the Kd of the interaction with IL8. These details have been obtained using the GRAMM software. A full and detailed explanation of how this tool was used may be found on our dry lab page, and we encourage any users of this part to go through the extensive documentation there. We will post a few images of the binding parameters obtained below:

  • il8nbdata.jpg
 Figure 2. The binding parameters obtained from the GRAMM software
  • il8nbold.jpg
 Figure 3: The structure of the RNA of the CDS produced on IVT of this sequence. 'Loss' function value (AUP) = 130.82000000000005
  • scaleaup.jpg
 Scale of the AUP

For more information on the AUP, please check out out pages of BBa_K4829019 onwards


References

  • Yue Wang, Rui Cheng Xu, Xiao Lei Zhang, Xiu Long Niu, Ye Qu, Ling Zhi Li, Xiang Yan Meng,Interleukin-8 secretion by ovarian cancer cells increases anchorage-independent growth, proliferation, angiogenic potential, adhesion and invasion,Cytokine,Volume 59, Issue 1,2012,Pages 145-155,ISSN 1043-4666
  • Yin, J., Zeng, F., Wu, N. et al. Interleukin-8 promotes human ovarian cancer cell migration by epithelial–mesenchymal transition induction in vitro. Clin Transl Oncol 17, 365–370 (2015). https://doi.org/10.1007/s12094-014-1240-4
  • Suyun Huang; Jubilee B. Robinson; Ariel DeGuzman; Corazon D. Bucana; Isaiah J. Fidler. Blockade of Nuclear Factor-κB Signaling Inhibits Angiogenesis and Tumorigenicity of Human Ovarian Cancer Cells by Suppressing Expression of Vascular Endothelial Growth Factor and Interleukin 8. Cancer Res (2000) 60 (19): 5334–5339.
  • J. Fujimoto, I. Aoki, S. Khatun, H. Toyoki, T. Tamaya. Clinical implications of expression of interleukin-8 related to myometrial invasion with angiogenesis in uterine endometrial cancers. Gynecologic tumors. https://doi.org/10.1093/annonc/mdf078.
  • Malihe Azadehrah, Shohre Vosoogh, Mahboobeh Azadehrah,The roles and therapeutic applications of cytokines in endometrial cancer,Journal of Reproductive Immunology,Volume 152,2022,103652,ISSN 0165-0378,https://doi.org/10.1016/j.jri.2022.103652.
  • RAZVAN CIORTEA1, DAN MIHU1 and CARMEN MIHAELA MIHU2. Association Between Visceral Fat, IL-8 and Endometrial Cancer. ANTICANCER RESEARCH 34: 379-384 (2014).
  • Lauren Ewington, Alexandra Taylor, Ruethairat Sriraksa, Yoshiya Horimoto, Eric W.-F. Lam, Mona A. El-Bahrawy,The expression of interleukin-8 and interleukin-8 receptors in endometrial carcinoma,Cytokine,Volume 59, Issue 2,2012,Pages 417-422,ISSN 1043-4666,https://doi.org/10.1016/j.cyto.2012.04.036.
  • Fang Deng, Yaguang Weng, Xian Li, Teng Wang, Mengtian Fan, Qiong Shi,Overexpression of IL-8 promotes cell migration via PI3K-Akt signaling pathway and EMT in triple-negative breast cancer,Pathology - Research and Practice,Volume 216, Issue 4,2020,152902,ISSN 0344-0338,https://doi.org/10.1016/j.prp.2020.152902.
  • Kim, S., Lee, J., Jeon, M. et al. MEK-dependent IL-8 induction regulates the invasiveness of triple-negative breast cancer cells. Tumor Biol. 3
  • Alraouji, N. N. and Aboussekhra, A. (2020). Tocilizumab inhibits il‐8 and the proangiogenic potential of triple negative breast cancer cells. Molecular Carcinogenesis, 60(1), 51-59. https://doi.org/10.1002/mc.23270


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