Part:BBa_K3504023

Off Switch

NOTICE: Parts in our range for this season have been created as a part of our Phase I design of our project. These parts HAVE NOT been tested or characterized in the lab due to COVID-19-related precautionary measures. We have enriched our new parts pages with data from literature and results from our modeling and simulations. If you are intending on using this part or others in our range, please keep in mind these limitations and update these parts with data from your experimentation. Feel free to reach us at: igem.afcm@gmail.com for further inquiries.

Part Description

A composite of parts (BBa_I712004,BBa_K3504019,BBa_K3504008) Which form as a whole an off switch using mir-126 & FF4 Mirna (BBa_K3504012) as as an inputs for a NOR gate that could protect our replicon vaccine vectors from being attacked by innate immunity inside dendritic cells and act as a safety switch

Usage

In mammalian cells, we face a challenging issue of burden-mitigating circuits. where miRNA-iFFLs, like (miR-FF4) have shown promising solution for it. We used miRNA to design a potable circuit and for optimizing endogenous miRNA that enable us to tailor our circuit to a specific cell line, thereby overcoming the issue of burden-mitigating circuits. Our input siRNA-FF4 works by regulating the expression of L7Ae. The repressor is under the control of the replicon SGP and additionally contains four repeats of the FF4 target site in its 3′UTR. A separate co-transfected replicon encodes output EGFP with two repeats of the K-turn motif in the 5′UTR.

The miRNA-126 has an already established role in angiogenesis and cancer, but recently in the current issue of Nature immunology, it was reported by agudo and colleagues that this miRNA-126 plays a part in survival and function of plasmocytoid dendritic cells (pDCs). These pDCs express Toll-like receptors (TLRs) 7 and 9 inside the cell which once activated results in the production of large quantities of (anti-viral) type I interferon. So this indicated that pDCs have a central role in the immune response. These pDCs are modulated by the miRNA-126 through a pathway (VEGFR2) meaning there is a potential capacity to control pDCs homeostasis and execute new deliberations on the effects on the innate immunity when the vascular endothelial growth factor (VEGF) as a target for therapeutic commitments. In pDCs, miR126 targets a negative regulator of mTOR for degradation.

Since the miRNA-126 is is highly up regulated in the pDCs we inserted a target site in the circuit vector and by that the miRNA-126 binds to the vector before being phagocytosed by the pDCs. This will cause cleavage of the circuit by the near end by the degradation sequence inserted in the 3' prime of the miRNA126 target site, therefore the circuit would be cut and left open, accordingly this will impair the replication of this vector. Since the miRNA-126 is is highly up regulated in the pDCs we inserted a target site in the circuit vector and by that the miRNA-126 binds to the vector before being phagocytosed by the pDCs. This will cause cleavage of the circuit by the near end by the degradation sequence inserted in the 3' prime of the miRNA126 target site, therefore the circuit would be cut and left open, accordingly this will impair the replication of this vector.

This will eventually lead to the unrecognition of the vector by pDCs and by that the other vectors will not be phagocytosed or presented on the surface which prevents the activation of the innate immunity and that makes the circuit safe from the attack of the innate immunity.

Characterization

We characterized this part by structural modelling and simulation of interaction between ff4 and its binding site using ff4 as an input for a NOR gate that could protect our replicon vaccine vectors from being attacked by innate immunity inside dendritic cells and act as a safety switch

Figure 2.This figure shows the sbol format of this composite part as well as way in which a nor gate works.

Figure 1.These figures illustrate the RNA Fold structure of multiple MiRNAs comparing there stabilities by free energy thermodynamic and showing there ability to induce down regulation of the circuit and work as an off switch.

Figure 3.This figure illustrate the interaction between FF4 and its binding site that showed energy equal to -3.14kcal\mol.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]