發(fā)布時(shí)間：2018-10-12 08:28

【摘要】：大腸桿菌具有遺傳背景清楚、培養(yǎng)簡單和生長迅速等優(yōu)點(diǎn),是工業(yè)生物技術(shù)領(lǐng)域中最常用的宿主菌之一。建立快速有效且能同時(shí)改造基因組上多個(gè)位點(diǎn)的策略和方法,將有助于更好地優(yōu)化代謝網(wǎng)絡(luò)。傳統(tǒng)的Red同源重組技術(shù)在對大腸桿菌基因組進(jìn)行編輯后,即使消除了抗性標(biāo)簽也會(huì)在基因組上殘留FRT位點(diǎn)序列,殘留的序列會(huì)嚴(yán)重影響隨后的同源重組效率。而目前基于sacB基因的兩步篩選無痕同源重組技術(shù)存在假陽性率較高的問題。本文試圖利用lac阻遏系統(tǒng)的特點(diǎn),將lacI基因選為新的負(fù)篩選標(biāo)記,構(gòu)建一種新的兩步篩選無痕同源重組的技術(shù),降低了假陽性率。此無痕同源重組技術(shù)的建立,也有利于結(jié)合其他無痕同源重組技術(shù),實(shí)現(xiàn)大腸桿菌基因組上多位點(diǎn)的同時(shí)原位改造,對大腸桿菌菌株性狀的改良具有重要的意義。本文首先將增強(qiáng)型啟動(dòng)子啟動(dòng)的lacI基因作為負(fù)篩選標(biāo)記,并與作為正篩選標(biāo)記的抗性基因連接,獲得正負(fù)篩選標(biāo)記基因盒。此基因盒中的抗性基因可靈活選擇,這為與其他無痕同源重組技術(shù)聯(lián)用實(shí)現(xiàn)大腸桿菌基因組多位點(diǎn)同時(shí)原位改造提供了可能。本文接著以EcHW2f為初始菌,先敲除了其基因組中天然的lacI基因及其啟動(dòng)子,再將基因組上kan抗性基因的天然啟動(dòng)子替換為lac啟動(dòng)子,完成本底菌的構(gòu)建。使得前期構(gòu)建的正負(fù)篩標(biāo)記選基因盒中的lacI基因成為負(fù)篩選標(biāo)記的唯一供體,在進(jìn)行負(fù)篩選前阻遏本底菌中kan抗性基因的表達(dá)。在此基礎(chǔ)上,利用本lacI兩步篩選無痕同源重組系統(tǒng),將talB基因的天然啟動(dòng)子,成功替換為人工啟動(dòng)子M1-93,驗(yàn)證了本技術(shù)的有效性。通過一系列的對照試驗(yàn),探索確定了 250mg/l的卡那霉素作為負(fù)篩選時(shí)使用的最優(yōu)篩選濃度,使得假陽性率相比cat-sacB系統(tǒng)的20%,降低到了 4%,大大提高了篩選效率。啟動(dòng)子替換后該菌株的番茄紅素單位產(chǎn)量提高了 23%。本文最后在常用的基因工程宿主DH5a中,通過將Plac-lacO-kan基因序列同源替換基因組中的lacI基因,一次重組就完成了本底菌的構(gòu)建,從而在DH5a中也可使用此系統(tǒng)實(shí)現(xiàn)基因組上的無痕同源重組。由此可見,其他的大腸桿菌宿主菌均可通過此簡單的操作完成本底菌的構(gòu)建,從而就可利用本文所構(gòu)建的無痕同源技術(shù)實(shí)現(xiàn)基因組上基因的快速改造�？傊�,本文致力于基于lac阻遏系統(tǒng)的特點(diǎn),構(gòu)建新的兩步重組方法,滿足無痕重組與可篩選的需求,有助于更好地通過同源重組的方式進(jìn)行代謝網(wǎng)絡(luò)的優(yōu)化,有望在對菌株性狀的改良中發(fā)揮重要的作用。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78

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本文編號(hào)：2265460