Difference between revisions of "Team:AFCM-Egypt/Design"
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<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #000000;"><span style="color: #ffffff;">Part</span> </span><span style="color: #ff9900;">BBa_K2217019</span><span style="color: #ffffff;"> was composed of CMV Enhancer as well as CMV Promoter (</span><span style="color: #ff9900;">BBa_K2217000</span> <span style="color: #000000;"><span style="color: #ffffff;">and </span> </span><span style="color: #ff9900;">BBa_K2217006</span><span style="color: #ffffff;"> respectively) as constitutive promoters to enhance the transcription process of the Cas9 (</span><span style="color: #ff9900;">BBa_K1218011</span><span style="color: #ffffff;">) part which was transcribed using T7 Promoter (</span><span style="color: #ff9900;">BBa_K2217007</span><span style="color: #ffffff;">) following Protocol<sup>[3]</sup>. 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 (</span><span style="color: #ff9900;">BBa_K2217009</span><span style="color: #ffffff;">) to be transfected on a separate donor vector then terminated with CYC1 terminator(</span><span style="color: #ff9900;">BBa_K2217008</span></span><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">).</span><span style="font-size: 14.0pt; line-height: 115%; font-family: 'Trebuchet MS','sans-serif'; color: #282828; background: white;"><br /><br /></span></p> | <p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #000000;"><span style="color: #ffffff;">Part</span> </span><span style="color: #ff9900;">BBa_K2217019</span><span style="color: #ffffff;"> was composed of CMV Enhancer as well as CMV Promoter (</span><span style="color: #ff9900;">BBa_K2217000</span> <span style="color: #000000;"><span style="color: #ffffff;">and </span> </span><span style="color: #ff9900;">BBa_K2217006</span><span style="color: #ffffff;"> respectively) as constitutive promoters to enhance the transcription process of the Cas9 (</span><span style="color: #ff9900;">BBa_K1218011</span><span style="color: #ffffff;">) part which was transcribed using T7 Promoter (</span><span style="color: #ff9900;">BBa_K2217007</span><span style="color: #ffffff;">) following Protocol<sup>[3]</sup>. 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 (</span><span style="color: #ff9900;">BBa_K2217009</span><span style="color: #ffffff;">) to be transfected on a separate donor vector then terminated with CYC1 terminator(</span><span style="color: #ff9900;">BBa_K2217008</span></span><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">).</span><span style="font-size: 14.0pt; line-height: 115%; font-family: 'Trebuchet MS','sans-serif'; color: #282828; background: white;"><br /><br /></span></p> | ||
<p style="text-align: center;"> </p> | <p style="text-align: center;"> </p> | ||
| − | <p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><img src="https://static.igem.org/mediawiki/2017/c/c8/AFCM-Egypt-Design7%268.png" | + | <p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><img src="https://static.igem.org/mediawiki/2017/c/c8/AFCM-Egypt-Design7%268.png" style="width:100%" /></span><span style="font-size: 14.0pt; line-height: 115%; font-family: 'Trebuchet MS','sans-serif'; color: #282828; background: white;"><br /></span></p> |
<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #ffffff;">Fig-3 Composite Part BBa_K2217020</span><br /></span></p> | <p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #ffffff;">Fig-3 Composite Part BBa_K2217020</span><br /></span></p> | ||
<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #ffffff;">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</span> <span style="color: #ffffff;">(</span><span style="color: #ff9900;">BBa_K2217013</span><span style="color: #ffffff;">) while circular RNA (hsa-circ-0000064) was submitted to the registry after biomarker filtration using bioinformatics databases demonstrated at our </span></span><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">biomarker filtration</span> <span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"> page</span></span><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #ffffff;"> (</span><span style="color: #ff9900;">BBa_K2217001</span><span style="color: #ffffff;">). Finally it was terminated by SV40 poly(A) signal Termination (</span><span style="color: #ff9900;">BBa_K2217005</span><span style="color: #ffffff;">).</span></span></p> | <p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #ffffff;">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</span> <span style="color: #ffffff;">(</span><span style="color: #ff9900;">BBa_K2217013</span><span style="color: #ffffff;">) while circular RNA (hsa-circ-0000064) was submitted to the registry after biomarker filtration using bioinformatics databases demonstrated at our </span></span><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">biomarker filtration</span> <span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"> page</span></span><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #ffffff;"> (</span><span style="color: #ff9900;">BBa_K2217001</span><span style="color: #ffffff;">). Finally it was terminated by SV40 poly(A) signal Termination (</span><span style="color: #ff9900;">BBa_K2217005</span><span style="color: #ffffff;">).</span></span></p> | ||
Latest revision as of 00:22, 27 October 2017
Biomarker Filtration
Steps Of Our Biomarker filtration
Design
Our design aims to compare the function of both circuits at regulating non-coding RNAs
| CRISPR circuit | ceRNA circuit |
|
BBa_K2217026 |
BBa_K2217025 |
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 syntheticTM Language using cytostudio.[7]
References
[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
[7] https://moleculamaxima.com/
