Difference between revisions of "Part:BBa K3504019"

Latest revision as of 21:25, 26 October 2020

FF4 Binding Site

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

Binding Site for MiRNA FF4, which is a micro RNA that has the ability to repress transcription. MiRNA FF4 is one of microRNAs that were constructed according to the pPRIME approach with stem-loops of functional sequence cloned into miR-30 backbone and fused into 3’-UTR of CMV-driven neomycin resistance gene. Their names are identical to their parent siRNAmolecules, i.e., FF3, FF4, FF5 and FF6

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.

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 all which was used to characterize the part BBa_K511101.

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

Figure 1.This figure illustrate the interaction between FF4 and its binding site that showed energy equal to -3.14kcal\mol.

References

1-Wroblewska, L., Kitada, T., Endo, K., Siciliano, V., Stillo, B., Saito, H., & Weiss, R. (2015, August). Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery. Retrieved October 26, 2020, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4532950/

Sequence and Features