Part:BBa_K4808003
g-rhtA
The g-rhtA is a guide RNA that can form a complex with Cas 9 in E.coli cicc 20905. It is a specific RNA sequence (around 20 bp) that recognizes the rhtA gene and directs the Cas 9 protein there for gene knocking out.
This year, our AIS-China successfully achieved the knockout of three genes on E.coli, CICC20905, a strain that has not been used in igem and has been studied less. we provide a complete set of guidelines for the use of gene knockouts for future igemers. Our contribution parts include three specific gRNA, pTargat plasmids, pEcCas plasmids needed to knock out genes. The collection is: BBa_K4808003、BBa_K4808004、BBa_K4808005、BBa_K4808009 、BBa_K4808011.
Characterization
CRISPR-CAS 9
CRISPR-Cas 9 systems contain two components: a guide RNA and a Cas 9 protein. The gRNA is a short synthetic RNA that defines the genomic target to be modified and the Cas9 protein is a DNA endonuclease. These two components will form a complex which recognizes the PAM site and binds to the target site, and then uses the cas9 protein to cut the double-stranded DNA in the target region to break it, inducing cells to perform homologous recombination repair to achieve the purpose of gene knockout or knockin.
It is one of the most scalable genome editing technologies due to the ease of generating gRNAs. Simply changing the target sequence present in the gRNA can alter the genomic target of the Cas protein thus enabling the knockout of different genes.
Our knockout method
Step 1: Preparation of competent cells and pEcCas transformation Transfer pEcCas into competent cells of the target strain through chemical transformation
Step 2: Construct pTartget plasmid and obtain donor DNA pTarget plasmid: Ncbi obtains the gene sequence to be knocked out, and chopchop is used as a tool to find gRNA, which is generally 20 bp. The 20 bp gRNA was assembled by Gibson to construct the pTarget plasmid. Donor DNA: Design and produce donor DNA containing homologous arms. Let E. coli perform homologous recombination repair, construct upDNA and downDNA through one round of PCR, and assemble upDNA and downDNA through two rounds of PCR to construct donor DNA.
Step 3: Place ptarget and donor simultaneously into E. coli in step 1 through electroporation to achieve knockout of the target gene. (See protocol for methods)
Step 4: Curing plasmid Positive clones with successful knockout were identified by colony PCR. a. To cure the pTarget plasmid, we put the positive clone into LB liquid medium containing rhamnose and kanamycin, culture it for 6 hours, and then transfer it to the medium without antibody at a ratio of 1:100; b. Curing for pEcCas plasmid, we add sucrose to the liquid culture medium, culture it overnight, and streak it onto the LB plate containing sucrose without antibody the next day; c. Screen the colonies on LB plates with kanamycin, LB plates with spectinomycin and LB plates, colonies that grew only on LB plates were cured both pEcCas and pTarget.
Our achievement
rhtA gene encodes a strong threonine exporter and transfers threonine out of the cell. The gene rhtA knockout resulted in the reduction of extracellular secretion of threonine so there are more amount of threonine can be kept inside the cell for the further production of a-KB. We design the pTarget plasmid that carrying specific gRNA sequence which can identity the rhtA gene, then we obtained donorDNA through two rounds of PCR. The donorDNA was used for homologous recombination with genomic DNA. We then put this pTarget plasmid and donor DNA into AIS-0 strains that has already carried pEcCas plasmid for the CRISPR-CAS 9 knockout experiment. (We referred to the experimental procedures published by Qi Li, Bingbing Sun, et al. in 2020) Through the results of colony PCR and gene sequencing, we confirmed the successful knockout of rhtA.
Figure 1: （A）the design of pEcCas、pTarget plasmid and donorDNA for gene knockout (B) verified the construction of pTarget-g-rhtA result through the sequencing testing. (C) colony PCR to respectively determine the knock-out of rhtA (D) verified the knock-out result through the sequencing testing
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
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- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References：
Cheng L, Wang J, Zhao X, et al. An antiphage Escherichia coli mutant for higher production of L-threonine obtained by atmospheric and room temperature plasma mutagenesis. Biotechnol Prog. 2020;36(6):e3058. doi:10.1002/btpr.3058
Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021;53(5):620-627. doi:10.1093/abbs/gmab036
Restrepo-Pineda S, O Pérez N, Valdez-Cruz NA, Trujillo-Roldán MA. Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights. FEMS Microbiol Rev. 2021;45(6):fuab023. doi:10.1093/femsre/fuab023
Chen L, Chen Z, Zheng P, Sun J, Zeng AP. Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli. Appl Microbiol Biotechnol. 2013;97(7):2939-2949. doi:10.1007/s00253-012-4176-z
Zhang C, Qi J, Li Y, et al. Production of α-ketobutyrate using engineered Escherichia coli via temperature shift. Biotechnol Bioeng. 2016;113(9):2054-2059. doi:10.1002/bit.25959
Park JH, Oh JE, Lee KH, Kim JY, Lee SY. Rational design of Escherichia coli for L-isoleucine production. ACS Synth Biol. 2012;1(11):532-540. doi:10.1021/sb300071a
Hao R, Wang S, Jin X, Yang X, Qi Q, Liang Q. Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine. Front Bioeng Biotechnol. 2023;11:1118948. Published 2023 Mar 2. doi:10.3389/fbioe.2023.1118948
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