RNA

Part:BBa_K2924007

Designed by: Vanessa Valencia   Group: iGEM19_Duesseldorf   (2019-10-14)
Revision as of 13:48, 20 October 2019 by AndreasN (Talk | contribs)


guideRNA from enoyl-[acyl-carrier-protein] reductase

fabI guide RNA of Synechocystis sp.

Usage and Biology

Fig. 1: Position of sgRNA (orange) in the Enoyl-[acyl-carrier-protein] reductase gene.
Fig. 2: Reaction scheme of the formation of acyl-[acyl-carrier-protein] by reductase

This part contains the enoyl-[acyl-carrier-protein] reductase guide RNA of Synechocystis sp. PCC 6803. It was used for an induced knock-down of fabI with a CRISPRi/dCas9-system, which was kindly provided by Yao et al. (2015)1. The Enoyl-[acyl-carrier-protein] reductase can be found under the UniProt ID: FABI_SYNY32, and is involved in fatty acid biosynthesis and metabolism3. The gene is positioned in the genome at 503883- 504719 (837 bp) bases3. The guide RNA was obtained by using the CRISPR guide from benchling4. The sgRNA in the gene is located at 55-74 bp in the + strand (Fig. 1). The sequence of the sgRNA is aattctatgttagatctcag , has anOn-Target Score of 73.1 and an Off-Target Score of 49.9.

Enoyl-[acyl-carrier-protein] reductase is a key enzyme of the fatty acid synthesis system. It catalyzes the NAD-dependent reduction of a carbon-carbon double bond in trans-2,3-dehydroacyl-[acyl-carrier-protein] to acyl-[acyl-carrier-protein] 5, 6(Fig. 2). In overall, this enzyme is involved in the elongations steps of the fatty acid synthesis, which is used for producing membrane lipids, lipid A, etc 6.

The short guide RNA (sgRNA) has been cloned into a vector containing a neutral site of Synechocystis sp. PCC 6803. That is a homologous sequence of its genome to ensure a knock-in into the genome (Fig. 3)1.

Due to the created knock-in containing a resistance for antibiotic in close proximity to the sgRNA, the target enzyme can down-regulated with a CRISPRi/dCas9 - system 1. This system is induced by anhydrotetracycline (aTc) that activates the synthesis of the dCas9, which then binds to the sgRNA. The formed complex is able to bind complementary to the targeted gene and stops the transcription of it (Fig. 4).

Fig. 3: Scheme of a knock-in as a consequence of homologous recombination in Synechocystis.
Fig. 4: Scheme of function of the CRISPRi/dCas9 - system. The dCas9 (yellow) binds with the sgRNA to the complementary DNA strand and inhibits the transcription by RNA polymerase II (blue).



















Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 12
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None