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首页> 外文期刊>Microbial Cell Factories >Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci
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Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci

机译：敲入/敲除（KIKO）载体，用于将大型DNA序列（包括整个代谢途径）快速整合到特征明确的基因座上的大肠杆菌染色体上

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Background Metabolic engineering projects often require integration of multiple genes in order to control the desired phenotype. However, this often requires iterative rounds of engineering because many current insertion approaches are limited by the size of the DNA that can be transferred onto the chromosome. Consequently, construction of highly engineered strains is very time-consuming. A lack of well-characterised insertion loci is also problematic. Results A series of knock-in/knock-out (KIKO) vectors was constructed for integration of large DNA sequences onto the E. coli chromosome at well-defined loci. The KIKO plasmids target three nonessential genes/operons as insertion sites: arsB (an arsenite transporter); lacZ (β-galactosidase); and rbsA-rbsR (a ribose metabolism operon). Two homologous ‘arms’ target each insertion locus; insertion is mediated by λ Red recombinase through these arms. Between the arms is a multiple cloning site for the introduction of exogenous sequences and an antibiotic resistance marker (either chloramphenicol or kanamycin) for selection of positive recombinants. The resistance marker can subsequently be removed by flippase-mediated recombination. The insertion cassette is flanked by hairpin loops to isolate it from the effects of external transcription at the integration locus. To characterize each target locus, a xylanase reporter gene (xynA) was integrated onto the chromosomes of E. coli strains W and K-12 using the KIKO vectors. Expression levels varied between loci, with the arsB locus consistently showing the highest level of expression. To demonstrate the simultaneous use of all three loci in one strain, xynA, green fluorescent protein (gfp) and a sucrose catabolic operon (cscAKB) were introduced into lacZ, arsB and rbsAR respectively, and shown to be functional. Conclusions The KIKO plasmids are a useful tool for efficient integration of large DNA fragments (including multiple genes and pathways) into E. coli. Chromosomal insertion provides stable expression without the need for continuous antibiotic selection. Three non-essential loci have been characterised as insertion loci; combinatorial insertion at all three loci can be performed in one strain. The largest insertion at a single site described here was 5.4 kb; we have used this method in other studies to insert a total of 7.3 kb at one locus and 11.3 kb across two loci. These vectors are particularly useful for integration of multigene cassettes for metabolic engineering applications.
机译：背景技术代谢工程项目通常需要整合多个基因以控制所需的表型。但是，由于许多当前的插入方法受到可转移到染色体上的DNA大小的限制，因此这通常需要反复进行工程设计。因此，构建高度工程化的菌株非常耗时。缺乏特征明确的插入位点也是有问题的。结果构建了一系列敲入/敲除（KIKO）载体，用于将大DNA序列整合到明确定义的基因座上的大肠杆菌染色体上。 KIKO质粒靶向三个非必需基因/操纵子作为插入位点：arsB（亚砷转运蛋白）; lacZ（β-半乳糖苷酶）;和rbsA-rbsR（核糖代谢操纵子）。每个插入基因座均靶向两个同源的“臂”。通过这些臂的λRed重组酶介导插入。在两臂之间是用于导入外源序列的多个克隆位点，以及用于选择阳性重组体的抗生素抗性标记（氯霉素或卡那霉素）。随后可以通过脂肪酶介导的重组去除抗性标记。插入盒的侧面有发夹环，可将其与整合位点处的外部转录隔离。为了表征每个靶基因座，使用KIKO载体将木聚糖酶报道基因（xynA）整合到大肠杆菌菌株W和K-12的染色体上。表达水平在基因座之间变化，而arsB基因座始终显示最高表达水平。为了证明在一个菌株中同时使用所有三个基因座，分别将xynA，绿色荧光蛋白（gfp）和蔗糖分解代谢操纵子（cscAKB）引入了lacZ，arsB和rbsAR中，并证明它们是有功能的。结论KIKO质粒是将大的DNA片段（包括多个基因和途径）有效整合到大肠杆菌中的有用工具。染色体插入无需连续抗生素选择即可提供稳定的表达。三个非必需基因座已被定性为插入基因座。可以在一个菌株中在所有三个基因座处进行组合插入。此处描述的单个位点的最大插入片段为5.4 kb;我们已在其他研究中使用此方法在一个基因座插入总共7.3 kb，在两个基因座插入了11.3 kb。这些载体对于用于代谢工程应用的多基因盒的整合特别有用。

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