基于Lac阻遏系统和抗性筛选实现大肠杆菌基因无痕同源重组的研究

发布时间：2018-10-12 08:28

【摘要】：大肠杆菌具有遗传背景清楚、培养简单和生长迅速等优点,是工业生物技术领域中最常用的宿主菌之一。建立快速有效且能同时改造基因组上多个位点的策略和方法,将有助于更好地优化代谢网络。传统的Red同源重组技术在对大肠杆菌基因组进行编辑后,即使消除了抗性标签也会在基因组上残留FRT位点序列,残留的序列会严重影响随后的同源重组效率。而目前基于sacB基因的两步筛选无痕同源重组技术存在假阳性率较高的问题。本文试图利用lac阻遏系统的特点,将lacI基因选为新的负筛选标记,构建一种新的两步筛选无痕同源重组的技术,降低了假阳性率。此无痕同源重组技术的建立,也有利于结合其他无痕同源重组技术,实现大肠杆菌基因组上多位点的同时原位改造,对大肠杆菌菌株性状的改良具有重要的意义。本文首先将增强型启动子启动的lacI基因作为负筛选标记,并与作为正筛选标记的抗性基因连接,获得正负筛选标记基因盒。此基因盒中的抗性基因可灵活选择,这为与其他无痕同源重组技术联用实现大肠杆菌基因组多位点同时原位改造提供了可能。本文接着以EcHW2f为初始菌,先敲除了其基因组中天然的lacI基因及其启动子,再将基因组上kan抗性基因的天然启动子替换为lac启动子,完成本底菌的构建。使得前期构建的正负筛标记选基因盒中的lacI基因成为负筛选标记的唯一供体,在进行负筛选前阻遏本底菌中kan抗性基因的表达。在此基础上,利用本lacI两步筛选无痕同源重组系统,将talB基因的天然启动子,成功替换为人工启动子M1-93,验证了本技术的有效性。通过一系列的对照试验,探索确定了 250mg/l的卡那霉素作为负筛选时使用的最优筛选浓度,使得假阳性率相比cat-sacB系统的20%,降低到了 4%,大大提高了筛选效率。启动子替换后该菌株的番茄红素单位产量提高了 23%。本文最后在常用的基因工程宿主DH5a中,通过将Plac-lacO-kan基因序列同源替换基因组中的lacI基因,一次重组就完成了本底菌的构建,从而在DH5a中也可使用此系统实现基因组上的无痕同源重组。由此可见,其他的大肠杆菌宿主菌均可通过此简单的操作完成本底菌的构建,从而就可利用本文所构建的无痕同源技术实现基因组上基因的快速改造。总之,本文致力于基于lac阻遏系统的特点,构建新的两步重组方法,满足无痕重组与可筛选的需求,有助于更好地通过同源重组的方式进行代谢网络的优化,有望在对菌株性状的改良中发挥重要的作用。
[Abstract]:E. coli is one of the most commonly used host bacteria in the field of industrial biotechnology because of its advantages of clear genetic background, simple culture and rapid growth. To establish a rapid and effective strategy and method for simultaneous modification of multiple loci in the genome will contribute to better optimization of metabolic networks. After editing the genome of Escherichia coli, the traditional Red homologous recombination technique will leave the FRT site sequence on the genome even if the resistance tag is eliminated, and the residual sequence will seriously affect the efficiency of the subsequent homologous recombination. At present, two-step screening method based on sacB gene has the problem of high false positive rate. Based on the characteristics of lac repressor system, this paper attempts to select lacI gene as a new negative screening marker and construct a new two-step screening technique for homologous recombination without trace, which reduces the false positive rate. The establishment of this traceless homologous recombination technique is also beneficial to the realization of simultaneous in situ transformation of multiple loci in Escherichia coli genome, which is of great significance to the improvement of E. coli strain traits. In this paper, the lacI gene initiated by the enhanced promoter was first used as a negative screening marker, and the positive and negative screening marker gene box was obtained by ligating with the resistance gene as a positive screening marker. The resistance genes in this gene box can be selected flexibly, which makes it possible to realize simultaneous in situ transformation of Escherichia coli genome with other non-trace homologous recombination techniques. In this paper, EcHW2f was used as the initial strain, the natural lacI gene and its promoter were knocked out, and then the natural promoter of the kan resistance gene was replaced with the lac promoter to complete the construction of the background bacterium. The lacI gene in the positive and negative sieve marker box was the only donor of negative screening marker, and the expression of kan resistance gene was inhibited before negative screening. On this basis, the lacI two-step screening homologous recombination system was used to replace the natural promoter of talB gene with the artificial promoter M1-93, which proved the effectiveness of this technique. Through a series of controlled experiments, the optimal screening concentration of kanamycin for negative screening of 250mg/l was determined. The false positive rate was reduced to 4 parts compared with 20 in cat-sacB system, and the screening efficiency was greatly improved. The lycopene unit yield of the strain increased by 23% after promoter replacement. Finally, in the common genetic engineering host DH5a, by replacing the lacI gene in the genome with the homologous sequence of the Plac-lacO-kan gene, we completed the construction of the background bacteria in a single recombination. This system can also be used in DH5a to realize the homologous recombination of the genome. It can be seen that other Escherichia coli host bacteria can complete the construction of the background bacteria through this simple operation, so that the rapid transformation of genes on the genome can be realized by using the non-trace homology technology constructed in this paper. In a word, based on the characteristics of lac repressor system, a new two-step recombination method is constructed to meet the needs of traceless recombination and sieving, which is helpful to optimize metabolic network through homologous recombination. It is expected to play an important role in the improvement of strain traits.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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