Difference between revisions of "Team:AFCM-Egypt/Applied Design"

Biomarker Filtration

Steps Of Our Biomarker filtration 

Design

Our design aims to compare the function of both circuits at regulating non-coding RNAs

Fig-1 Composite Part BBa_K2217018

Part BBa_K2217018 was composed of  U6 (BBa_K2217002) as a promoter for gRNA (BBa_K2217003)(Designed using MIT CRISPR web tool according to the addgene protocol).^[1][2] The gRNA needs a scaffold to guide the Cas9 (BBa_K2217004) and finally it was terminated by SV40 poly(A) signal Termination (BBa_K2217005).

Fig-2 Composite Part BBa_K2217019

Part BBa_K2217019 was composed of CMV Enhancer as well as CMV Promoter (BBa_K2217000 and  BBa_K2217006 respectively) as constitutive promoters to enhance the transcription process of the Cas9 (BBa_K1218011) part which was transcribed using  T7 Promoter (BBa_K2217007) following Protocol^[3]. The design aimed at generating Homology Directed Repair instead of Non-Homologous end Joining repair of Cas9 by knocking in the circular RNA as a Competing endogenous RNA enhancing its transcription to regulate miRNA action, so we designed Homology repair template (BBa_K2217009) to be transfected on a separate donor vector then terminated with CYC1 terminator(BBa_K2217008).

Fig-3 Composite Part BBa_K2217020

In order to enhance transcription of the circular RNA as a competing endogenous RNA in a ceRNA network, we used the CAG promoter composed of CMV enhancer and Chicken B-actin Promoter (BBa_K2217013) while circular RNA (hsa-circ-0000064) was submitted to the registry after biomarker filtration using bioinformatics databases demonstrated at our biomarker filtration  page (BBa_K2217001). Finally it was terminated by SV40 poly(A) signal Termination (BBa_K2217005).

 



Fig-4 Composite Part BBa_K2217022

Part BBa_K2217022 was composed of CMV Enhancer as well as CMV Promoter (BBa_K2217000 and  BBa_K2217006 respectively) as constitutive promoters to enhance the transcription process of the laci that was submitted (Ba_K2217012). We also improved the fragment’s characterization by adding the miRNA binding site of the miRNA mir-1825 as determined computationally using circ interactome database ^[4] where we hypothesised its function as a trigger for miRNA binding and synthesis. Finally, it was terminated by SV40 poly(A) signal Termination (BBa_K2217005).

Fig-5 Composite Part BBa_K2217023

Part BBa_K2217023 is composed of lac operator (BBa_K2217016) and the lac promoter (BBa_K2217017) as a weak constitutive promoter in a trial to improve characterization of ceRNA network using yfb (BBa_K2217014) detection using flow cytometry. Finally, it was terminated by Poly_gh termination (BBa_K2217015).

All Parts were designed in SBOL format using SBOLDesigner.^[5] Sequence editing was performed on Benchling^[6] and composites were constructed in synthetic^TM Language using cytostudio.^[7]

References

[1]  http://crispr.mit.edu

[2]  https://media.addgene.org/cms/files/hCRISPR_gRNA_Synthesis.pdf

[3] Romanienko PJ, Giacalone J, Ingenito J, et al. A Vector with a Single Promoter for In Vitro Transcription and Mammalian Cell Expression of CRISPR gRNAs. Fujii H, ed. PLoS ONE. 2016;11(2):e014836.

[4] Dudekula DB, Panda AC, Grammatikakis I, De S, Abdelmohsen K, Gorospe M. CircInteractome: A web tool for exploring circular RNAs and their interacting proteins and microRNAs. RNA Biology. 2016;13(1):34-42.

[5] www.async.ece.utah.edu/SBOLDesigner

[6] https://benchling.com/

[7] https://moleculamaxima.com/