Difference between revisions of "Part:BBa K4139002:Design"
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===Design Notes=== | ===Design Notes=== | ||
| − | In order to determine which region of the mcyH gene our crRNA should target, we used the RNAfold tool. This allowed us to locate a target sequence that met the ideal design criteria of being close to the 5' end of the sequence, being relatively unfolded region in the mRNA secondary structure, and having a high probability of remaining single-stranded | + | In order to determine which region of the mcyH gene our crRNA should target, we used the RNAfold tool. This allowed us to locate a target sequence that met the ideal design criteria of being close to the 5' end of the sequence, being relatively unfolded region in the mRNA secondary structure, and having a high probability of remaining single-stranded. |
| + | This crRNA coding region was designed to be the reverse compliment of the region it will target. | ||
| + | Note that this crRNA will target E.coli that has been transformed with the mcyH gene that has been codon-optimized for E.coli, rather than the mcyH gene from the Microcystis aeruginosa genome. | ||
===Source=== | ===Source=== | ||
Latest revision as of 03:14, 12 October 2022
mcyH crRNA for Lbu Cas13a (for E.coli)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 42
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
In order to determine which region of the mcyH gene our crRNA should target, we used the RNAfold tool. This allowed us to locate a target sequence that met the ideal design criteria of being close to the 5' end of the sequence, being relatively unfolded region in the mRNA secondary structure, and having a high probability of remaining single-stranded.
This crRNA coding region was designed to be the reverse compliment of the region it will target.
Note that this crRNA will target E.coli that has been transformed with the mcyH gene that has been codon-optimized for E.coli, rather than the mcyH gene from the Microcystis aeruginosa genome.
Source
This part was designed using a segment of the mcyH gene as a reference (GenBank: AAF00956.1)
References
Abudayyeh OO, Gootenberg JS, Konermann S, Joung J, Slaymaker IM, Cox DB, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV, Zhang F. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science. 2016 Aug 5;353(6299):aaf5573. doi: 10.1126/science.aaf5573. Epub 2016 Jun 2. PMID: 27256883; PMCID: PMC5127784.
Gruber AR, Lorenz R, Bernhart SH, Neuböck R, Hofacker IL. The Vienna RNA Websuite. Nucleic Acids Research, Volume 36, Issue suppl_2, 1 July 2008, Pages W70-W74, DOI: 10.1093/nar/gkn188
Lorenz, R. and Bernhart, S.H. and Höner zu Siederdissen, C. and Tafer, H. and Flamm, C. and Stadler, P.F. and Hofacker, I.L. "ViennaRNA Package 2.0", Algorithms for Molecular Biology, 6:1 page(s): 26, 2011
Mathews DH, Disney MD, Childs JL, Schroeder SJ, Zuker M, Turner DH. (2004) Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc Natl Acad Sci U S A 101(19):7287-92.
O'Connell MR. Molecular Mechanisms of RNA Targeting by Cas13-containing Type VI CRISPR–Cas Systems. Journal of Molecular Biology. 2019;431(1):66-87. doi: https://doi.org/10.1016/j.jmb.2018.06.029.
Pearson LA, Hisbergues M, Börner T, Dittmann E, Neilan BA. Inactivation of an ABC transporter gene, mcyH, results in loss of microcystin production in the cyanobacterium Microcystis aeruginosa PCC 7806. Appl Environ Microbiol. 2004;70(11):6370-8. Epub 2004/11/06. doi: 10.1128/aem.70.11.6370-6378.2004. PubMed PMID: 15528494; PubMed Central PMCID: PMCPMC525210.