CN114934057B - 一种适于大肠杆菌高效表达的IsDNA序列、制备方法、重组表达质粒及工程菌 - Google Patents
一种适于大肠杆菌高效表达的IsDNA序列、制备方法、重组表达质粒及工程菌 Download PDFInfo
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Abstract
本发明提供了的一种适于大肠杆菌高效表达的IsDNA序列、制备方法、重组表达质粒及工程菌,其表达产物具有高效催化吲哚促进靛蓝形成的作用;基于马蓝植株细胞色素P450mRNA序列(GenbankID:MG857655.1)的核酸和氨基酸序列信息,经过大肠杆菌(E.coli)密码子偏好优化获得;该IsDNA序列经化学合成后,运用亚克隆技术将合成的IsDNA序列插入到质粒pET‑22b中构建重组表达质粒,将重组表达质粒转化至大肠杆菌BL21(DE3),获得基因工程菌BL21(DE3)/IsDNA。该工程菌可在大肠杆菌中实现靛蓝色素生物合成,为后续应用生物合成技术实现靛蓝的工业化生产奠定基础。
Description
技术领域
本发明涉及生物技术、食品添加剂及染料制造技术领域,具体涉及一种适于大肠杆菌高效表达的IsDNA序列、制备方法、重组表达质粒及工程菌。
背景技术
靛蓝是色素中基本三原色之一,是一种重要的化工染料和食品色素添加剂。作为化工染料全球年需求量在8-80万吨之间,主要来源于化学合成。随着人们生活水平的提高和健康意识能力的增强,对于化学合成所需的有毒原料(苯胺、硝基苯、邻苯二甲酸酐等)及其产品均有较全面的了解,人们逐渐地认识到化学染料制成的织物产品对身体健康的危害。古老的靛蓝色素虽然在我国已经使用了2000多年了,且传统的提取工艺方法成熟。但是天然靛蓝来源极少,尽管能提取靛蓝的植物较多,如木蓝、菘蓝、蓼蓝、马蓝等,然而其靛蓝含量低,根本无法满足现代化工艺生产和市场的需要。寻找天然靛蓝的来源,优化和更新工艺提取方法,获得更多的天然靛蓝,以满足人们需要,既符合人类对天然、绿色、健康的追求目标,又符合全球生态保护、环境友好的发展理念。
现已发现,多种微生物在自然界可以合成靛蓝,如Pseudomonas sp.,Acinetobacter sp.,Bacillus megatreium等等。众多学者由此深受启发,如果能开发微生物合成靛蓝,这样就可以不受资源限制。如果能洞察靛蓝在微生物中的合成通路,分析靛蓝在微生物细胞中的合成机制,并以此对微生物进行人工改造,提高靛蓝的产量,就可以降低生产成本。运用微生物合成天然靛蓝色素,将成为淘汰化学合成有害色素的有效途径,是未来天然色素生产的发展趋势。目前,已有研究报道靛蓝色素是通过不同的酶作用于吲哚,使其发生氧化反应来实现,当该酶为单加氧酶时,其产物为靛蓝色素,当该酶为双加氧酶时,其产物为靛蓝和靛玉红混合色素。由于自然界中野生菌株产生靛蓝色素的量只是满足菌株自身生长的需要,因此靛蓝色素的生物转化量非常低。国内外学者通过分子生物学技术对不同的加氧酶(如萘双加氧酶、苯酚羟化酶、二甲苯单加氧酶、黄素蛋白单加氧酶、细胞色素P450单加氧酶、铜依赖单加氧酶等等)进行克隆,并构建工程菌进行合成靛蓝色素方面的研究,但其合成产量并不理想,只能说明通过加氧酶的克隆表达在细菌细胞内能合成靛蓝色素。尽管有些公司利用代谢工程的手段对合成途径及关键基因进行了优化改造,并取得了一定的效果,但终因资源、成本及技术等多方面因素的限制,微生物合成靛蓝工业化最终未能实现。因此,研究开发高产、稳定合成靛蓝的关键酶,构建高效合成靛蓝的工程菌,实现天然靛蓝的工业化合成,以满足人类生产生活及身体健康的需要,具有重大意义。
细胞色素P450单加氧酶广泛动物植物微生物中,在微生物合成靛蓝的研究中,以P450BM-3研究较多,普遍认为野生型的细胞色素P450单加氧酶不直接催化合成吲哚酚,大都是将细胞色素P450单加氧酶基因做了优化后才可以催化合成吲哚酚,进而形成靛蓝,目前也未见有细胞色素P450单加氧酶基因优化后序列的报道。关于植物中的细胞色素P450单加氧酶催化合成吲哚酚,进而形成靛蓝的研究,报道极少。
综上所述,急需一种能高产、稳定合成靛蓝的关键酶的、适于大肠杆菌高效表达的IsDNA序列、制备方法、重组表达质粒及工程菌以解决现有技术中存在的问题。
发明内容
本发明提供了一种适于大肠杆菌高效表达的IsDNA序列,具体方案如下:
一种适于大肠杆菌高效表达的IsDNA序列,所述IsDNA序列如SEQ ID NO.1所示。
本发明还提供一种IsDNA序列的制备方法,用于制备上述IsDNA序列,包括如下步骤:对Genbank ID为MG857655.1的马蓝细胞色素P450单加氧酶基因进行分析编码,并根据大肠杆菌的偏好性进行密码子偏好优化,得到IsDNA序列。
优选的,经大肠杆菌的偏好性进行密码子偏好优化的参数具体包括:
1)所述IsDNA序列的密码子适应指数CAI优化前为0.67,优化后为0.97;
2)所述IsDNA序列中的碱基GC含量优化前以及优化后均为0.5;
3)所述IsDNA序列优化前含有NcoI、XhoI、SacI和EcoR I限制性内切酶识别位点,优化后不含NcoI、XhoI、SacI和EcoR I限制性内切酶识别位点;所述IsDNA序列的回避序列优化后移除了2个AATAAA碱基序列和1个ATTAAA碱基序列;
4)删除所述IsDNA的重复序列;优化前重复序列的最大正向和最大反向长度均为12bp,优化后最大反向长度为14bp。
本发明还提供了一种表达IsDNA序列的重组表达质粒,含有上述IsDNA序列,所述表达IsDNA序列的重组表达质粒为pET-22b-IsDNA,得到所述表达IsDNA序列的重组表达质粒的过程为:选取质粒pET-22b;在IsDNA序列的两端分别加上限制性内切酶NdeI和HindIII位点识别序列5’-CATATG-3’和5’-AAGCTT-3’;使用限制性内切酶NdeI和HindIII对pET-22b进行双酶切;将双酶切后的pET-22b与IsDNA序列进行重组,得到表达IsDNA的重组表达质粒。
本发明还提供了一种表达IsDNA序列的工程菌,所述的工程菌含有上述IsDNA序列或重组表达质粒。
优选的,所述含有IsDNA序列的工程菌为BL21(DE3)/IsDNA,通过如下制备步骤制得:选取大肠杆菌BL21(DE3);利用热击转化方法将重组表达质粒转入大肠杆菌BL21(DE3)中,得到表达IsDNA的工程菌。
优选的,所述表达IsDNA的工程菌在生产天然的靛蓝色素中的应用。
应用本发明的技术方案,具有以下有益效果:
(1)本发明的IsDNA序列基于马蓝细胞色素P450单加氧酶基因序列,经过大肠杆菌(E.coli)密码子偏好优化所得。该基因序列能够在大肠杆菌中高效表达,进而可以实现天然的靛蓝色素的工业化生产。
(2)本发明的IsDNA序列是自然界中不存在的DNA序列,该序列能借助原核表达载体(质粒)实现在BL21(DE3)中高效表达产物,产物利用吲哚为底物催化合成天然靛蓝色素。本发明得到的工程菌株,为天然靛蓝色素生物合成的工业化定向设计奠定基础。
除了上面所描述的目的、特征和优点之外,本发明还有其它的目的、特征和优点。下面将参照图,对本发明作进一步详细的说明。
附图说明
构成本申请的一部分的附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。在附图中:
图1是本发明实施例中所述IsDNA序列密码子偏好调节前的示意图;
图2是本发明实施例中所述IsDNA序列密码子偏好调节后的示意图;
图3是本发明实施例中所述IsDNA序列碱基GC含量优化前的示意图;
图4是本发明实施例中所述IsDNA序列碱基GC含量优化后的示意图;
图5是本发明实施例中IsDNA序列和CYP450基因表达检测示意图;
图6是本发明实施例中BL21(DE3)/IsDNA与BL21(DE3)/CYP450工程菌株中相同条件下不同时间点(5h、8h、12h和24h)生产靛蓝色素的对比示意图。
具体实施方式
以下结合附图对本发明的实施例进行详细说明,但是本发明可以根据权利要求限定和覆盖的多种不同方式实施。
实施例1:适于大肠杆菌高效表达的DNA序列的设计与密码子偏好优化
为了提高马蓝细胞色素P450单加氧酶基因在大肠杆菌中的表达量,采用基因设计软件(OptimumGeneTM)对马蓝细胞色素P450单加氧酶基因(GenBank:MG857655.1)进行分析编码,并根据大肠杆菌偏好性进行密码子偏好优化,优化后得到序列命名为Indigosynthetic DNA序列,简称IsDNA序列,其序列如SEQ.ID.No.1所示。
参见图1和图2所示,所述DNA序列密码子偏好优化前后变化可见,参见图3和图4所述IsDNA序列中的GC含量保持不变为50%,但GC在序列中的分布位置发生了改变。
优化后得到的所述IsDNA序列的限制性内切酶与回避序列(Restriction EnzymeAnd Avoid Sequence)变化如表1和表2所示。
表1限制性内切酶位点变化
RestrictionName | Original | Optimization |
SacI | 1 | 0 |
EcoRI | 1 | 0 |
NcoI | 1 | 0 |
XhoI | 1 | 0 |
表2回避序列
Removed | Original | Optimization |
AATAAA | 2 | 0 |
ATTAAA | 1 | 0 |
具体的,适于大肠杆菌高效表达的IsDNA序列的设计与密码子偏好优化的具体过程如下:
1)Escherichia coli(大肠杆菌)密码子适应指数优化;
2)GC含量分布优化;
3)酶切位点的优化:为便于构建克隆及亚克隆,优化原有序列中NcoI、XhoI、SacI和EcoR I限制性内切酶识别序列;
4)IsDNA序列与马蓝细胞色素P450单加氧酶基因序列(GenBank:MG857655.1)的同源性仅为74.8%。由于优化前后DNA序列差异较大,在https://blast.ncbi.nlm.nih.gov/网站上不能比对上相关DNA序列(No significant similarity found),故命名为命名为Indigo synthetic DNA序列,简称IsDNA序列。
IsDNA序列如SEQ ID NO.1所示:
ATGGAAGCCACCTATGCAAGCGCAATCTATGGCGCAGTGGCACTGTTTCTGCTGTTTTATTATTATCTGCTGACCAAAAGCAGCAAACATAAACTGCCGCCGGAAGCCCCGGGTGCACGCCACTTACATCTGATGGCAGGTGGTGCAACCAGTAGCGCAAAACCGCCGCATATTATTCTGGGCGCCCTGAGTGATCAGCATGGCCCGATTTTTACCCTGCGCCTGGGTGTTCGCCGCATTCTGCTGGTGAGCAGCAGCCGTATTGCCAAAGAACTGTTTACCAGTAGTGATCTGGCCATTAGCAGCCGTCCGAAAACCCGTGGCATTAAGCATCTGGGCTATGATTTTGTTATGTTTGCATTTTCTCCGTATAGTGCATATTGGCGCCACATGCGTAAACTGGTTACCGTTGAACTGCTGAGCAGTCATCGTGCAGAACTGTTTAGCAGCGTGGGTATGGAAGAAGTGAAACAGAGTGTTAAAGAACTGCATGCAGTTTGGGAAGGCAAAAAAGATGGTAGCGGCCAGCTGCTGGTTGATATGCGTAATTGGCTGGCAGATATGAATCTGAATACCATTCTGCGTCAGGTGGTGGGCAAACGTCTGTGTGGTGGTGGCGGCGGTGACGATGCCGAAGAAATGCGCCAGTGTCGCGATGCAATTTGGGATTTCTTTCATCTGGTGGGCCTGTTTGTTCCGGCAGATGCACTGCCGTGGCTGGGCTGGCTGGATCTGGGTGGTTATGAAAAGAAAATGAAAGAAACCGCAAAGAAACTGGAAGGCATTATGGGCGGCTGGCTGGAAGAACATCGTCGCAAAGAATATAGTGGTGGTGAAGGTAAAGTGGAAGATTTTATGGATGTGATTCTGAGTGCAGTGCGCGGCAGTGAAGGTGAATATGAACATGATGTTGATACCGTGATTAAGAGCACCTGCCAGCTGATGATTCTGGGTGCAACCGATACCTTTGCCGTGACCCTGACCTGGGCCCTGAGCCTGCTGCTGAATAATCGTCATGTGCTGACCAAAGCCCAGGAAGAACTGGATAAACATGTGGGTCGTCATAAAGGTGTGAATAAGAGCGATATTTGCAATCTGGTTTATCTGCAGGCAATTGTGAAAGAAACCCTGCGTCTGTATCCGGCCGCCCCGCTGGGTGGTCCTCGTGAGTTTCGTGAAGATTGTAATATTGCCGGTTATCATATTCCGAAAGGTACCTGGCTGATGGTTAATGTTTGGAAACTGCATCGCGATCCGCAGGTGTGGCAGGATAATCCGCTGGATTTTAAACCGGAACGTTTTCTGACCACCCATAAAAATATGGATATTAATGGCCAGGATTTCGAACTGATGCCGTTTGGCGGCGGCCGTCGTATTTGCCCGGGCTTAAATCTGGGCATGCAGACCATTAATATGGTGCTGGCCAATCTGCTGCAGGCCTTTGAATTTGTTACCATTAATAATGAGGCGGTTGATATGACCGAAAGTGCAGGTCTGACCAATCTGAAAGCAACCCCGCTGGAAATTCTGATTACCCCGCGTCTGCCGCCGAATCTGTATTAA。
实施例2:表达IsDNA序列的重组表达质粒的获得以及表达马蓝细胞色素P450单加氧酶(CYP450)基因的重组表达质粒的获得
表达马蓝细胞色素P450单加氧酶(CYP450)基因的重组表达质粒的获得过程具体如下:选取质粒pET-22b,化学合成所述CYP450基因序列,在序列两端分别加上限制性内切酶NdeI和HindIII位点识别序列5’-CATATG-3’和5’-AAGCTT-3’,使用限制性内切酶NdeI和HindIII对质粒pET-22b进行双酶切,完成质粒的线性化,与化学合成的CYP450基因序列进行重组,构建pET-22b-CYP450重组表达质粒。
表达IsDNA序列的重组表达质粒的获得的具体过程如下:选取质粒pET-22b,化学合成所述IsDNA序列,在序列两端分别加上限制性内切酶NdeI和HindIII位点识别序列5’-CATATG-3’和5’-AAGCTT-3’,使用限制性内切酶NdeI和HindIII对质粒pET-22b进行双酶切,完成质粒的线性化,与化学合成IsDNA序列进行重组,构建pET-22b-IsDNA重组表达质粒。
实施例3:表达IsDNA序列的工程菌的获得以及达马蓝细胞色素P450单加氧酶(CYP450)基因的工程菌的获得
将表达IsDNA序列的重组表达质粒pET-22b-IsDNA和CYP450基因的重组表达质粒pET-22b-CYP450分别转化大肠杆菌BL21(DE3),分别制备成工程菌株BL21(DE3)/IsDNA和BL21(DE3)/CYP450。
具体的,BL21(DE3)感受态细胞的制备步骤如下:
1)挑取单菌落接种于LB培养基中,置于37℃摇床中,220rpm震荡培养过夜。
2)取2mL过夜培养物转接于200mL LB培养基中,在37℃摇床上剧烈振荡培养至OD600=0.6。
3)将菌液迅速置于冰上,在超净工作台和冰上操作以下步骤。
4)吸取1.5mL培养好的菌液至1.5mL离心管中,在冰上冷却10min。
5)4℃下4000rpm冷冻离心10min。
6)弃去上清,加入1.5mL冰冷的0.1mol/L的CaCl2溶液,用移液枪轻轻上下吸动打匀,使细胞重新悬浮。
7)4℃下4000rpm冷冻离心10min。
8)弃去上清,加入750μL冰冷的0.1mol/L的CaCl2溶液,用移液枪轻轻上下吸动打匀,使细胞重新悬浮。
9)4℃下4000rpm冷冻离心10min。
10)加入60μL冰冷0.1mol/L的CaCl2溶液(含15%甘油),用移液器轻轻上下吸动打匀,使细胞重新悬浮。
11)立即使用或迅速置于-70℃超低温保存。
具体的,所述的IsDNA序列和CYP450基因在大肠杆菌中过量表达,将转化后的平板中分别挑取BL21(DE3)/IsDNA和BL21(DE3)/CYP450单克隆,分别于LB培养基中过夜培养活化菌株,次日按1比100接种菌液到新鲜的培养基中,37℃培养约1-2h。待OD600值达到0.4-0.6之间,加入IPTG(终浓度为0.5mmol/L),于37℃诱导培养4h。取2mL菌液12000rmp离心2min,去除上清收集菌体,加200uL1×上样缓冲液,然后沸水煮5min,再12000rmp离心2min,取上清3uL进行上样电泳。电泳结束后用考马斯亮蓝染液染色,并用冰乙酸进行脱色处理,观察蛋白的表达。蛋白检测结果表明,BL21(DE3)/IsDNA和BL21(DE3)/CYP450工程菌株在IPTG的诱导下均可过量表达IsDNA蛋白和CYP450蛋白,如图5所示,其中:M为蛋白分子量标记;A为BL21(DE3)/CYP450,未诱导;B为BL21(DE3)/CYP450,IPTG诱导;C为BL21(DE3)/IsDNA,未诱导;D为BL21(DE3)/IsDNA,IPTG诱导。
具体的,工程菌株的培养方法具体如下:
1)将蛋白胨6-10g,酵母膏3-5g,氯化钠6-10g。
2)加无菌水配制成1000ml培养基(配制而成的培养基的pH值=7.0)。;
3)挑取新鲜培养的菌株接种于LB培养基中,置于恒温箱中37℃,150rpm培养24h进行活化。
4)将新鲜活化的种子培养物转接至装有100mL培养基的500mL锥形瓶中,分别设置添加吲哚与诱导剂进行对比,37℃,150rpm进行培养,依次在培养5小时、8小时、12小时和24小时时取培养液5ml于玻璃试管中,进行拍照,如图6所示。
5)收集50mL发酵液中的蓝色沉淀(含细菌),无菌水洗涤后超声破碎细胞,加入二甲基亚砜反复萃取靛蓝色素,过滤取滤液。
6)采用高效液相色谱仪检测靛蓝,检测条件为:C18色谱柱,二极管阵列检测器,流动相V(甲醇)∶V(超纯水)=9∶1,柱温30℃,流速0.2mL/min,检测波长610nm。
7)分别发酵5h、8h、12h和24h后,比较各菌株中靛蓝色素产量,参见图6,(其中:E号样品为:BL21(DE3)/CYP450;F号样品为:BL21(DE3)/IsDNA);可知BL21(DE3)/IsDNA明显高于与BL21(DE3)/CYP450靛蓝色素的含量。可见发明所提供的表达IsDNA序列的工程菌可应用于工业生产天然的靛蓝色素。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 湖南道生生物科技有限公司
<120> 一种适于大肠杆菌高效表达的IsDNA序列、制备方法、重组表达质粒及工程菌
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1563
<212> DNA
<213> IsDNA序列(人工合成)
<400> 1
atggaagcca cctatgcaag cgcaatctat ggcgcagtgg cactgtttct gctgttttat 60
tattatctgc tgaccaaaag cagcaaacat aaactgccgc cggaagcccc gggtgcacgc 120
cacttacatc tgatggcagg tggtgcaacc agtagcgcaa aaccgccgca tattattctg 180
ggcgccctga gtgatcagca tggcccgatt tttaccctgc gcctgggtgt tcgccgcatt 240
ctgctggtga gcagcagccg tattgccaaa gaactgttta ccagtagtga tctggccatt 300
agcagccgtc cgaaaacccg tggcattaag catctgggct atgattttgt tatgtttgca 360
ttttctccgt atagtgcata ttggcgccac atgcgtaaac tggttaccgt tgaactgctg 420
agcagtcatc gtgcagaact gtttagcagc gtgggtatgg aagaagtgaa acagagtgtt 480
aaagaactgc atgcagtttg ggaaggcaaa aaagatggta gcggccagct gctggttgat 540
atgcgtaatt ggctggcaga tatgaatctg aataccattc tgcgtcaggt ggtgggcaaa 600
cgtctgtgtg gtggtggcgg cggtgacgat gccgaagaaa tgcgccagtg tcgcgatgca 660
atttgggatt tctttcatct ggtgggcctg tttgttccgg cagatgcact gccgtggctg 720
ggctggctgg atctgggtgg ttatgaaaag aaaatgaaag aaaccgcaaa gaaactggaa 780
ggcattatgg gcggctggct ggaagaacat cgtcgcaaag aatatagtgg tggtgaaggt 840
aaagtggaag attttatgga tgtgattctg agtgcagtgc gcggcagtga aggtgaatat 900
gaacatgatg ttgataccgt gattaagagc acctgccagc tgatgattct gggtgcaacc 960
gatacctttg ccgtgaccct gacctgggcc ctgagcctgc tgctgaataa tcgtcatgtg 1020
ctgaccaaag cccaggaaga actggataaa catgtgggtc gtcataaagg tgtgaataag 1080
agcgatattt gcaatctggt ttatctgcag gcaattgtga aagaaaccct gcgtctgtat 1140
ccggccgccc cgctgggtgg tcctcgtgag tttcgtgaag attgtaatat tgccggttat 1200
catattccga aaggtacctg gctgatggtt aatgtttgga aactgcatcg cgatccgcag 1260
gtgtggcagg ataatccgct ggattttaaa ccggaacgtt ttctgaccac ccataaaaat 1320
atggatatta atggccagga tttcgaactg atgccgtttg gcggcggccg tcgtatttgc 1380
ccgggcttaa atctgggcat gcagaccatt aatatggtgc tggccaatct gctgcaggcc 1440
tttgaatttg ttaccattaa taatgaggcg gttgatatga ccgaaagtgc aggtctgacc 1500
aatctgaaag caaccccgct ggaaattctg attaccccgc gtctgccgcc gaatctgtat 1560
taa 1563
Claims (5)
1.一种适于大肠杆菌高效表达的IsDNA序列的制备方法,其特征在于,所述IsDNA序列如SEQ ID NO.1所示;
用于制备如上述所述的IsDNA序列,包括如下步骤:对Genbank ID为MG857655.1的马蓝细胞色素P450单加氧酶基因进行分析编码,并根据大肠杆菌的偏好性进行密码子偏好优化,得到IsDNA序列;
经大肠杆菌的偏好性进行密码子偏好优化的参数具体包括:
1)所述IsDNA序列的密码子适应指数CAI优化前为0.67,优化后为0.97;
2)所述IsDNA序列中的碱基GC含量优化前以及优化后均为0.5;
3)所述IsDNA序列优化前含有NcoI、XhoI、SacI和EcoR I限制性内切酶识别位点,优化后不含NcoI、XhoI、SacI和EcoR I限制性内切酶识别位点;所述IsDNA序列的回避序列优化后移除了2个AATAAA碱基序列和1个ATTAAA碱基序列;
4)删除所述IsDNA的重复序列;优化前重复序列的最大正向和最大反向长度均为12bp,优化后最大反向长度为14bp。
2.一种表达IsDNA序列的重组表达质粒,其特征在于,含有如权利要求1所述的IsDNA序列,所述表达IsDNA序列的重组表达质粒为pET-22b-IsDNA,得到所述表达IsDNA序列的重组表达质粒的过程为:选取质粒pET-22b;在IsDNA序列的两端分别加上限制性内切酶NdeI和HindIII位点识别序列5’-CATATG-3’和5’-AAGCTT-3’;使用限制性内切酶NdeI和HindIII对pET-22b进行双酶切;将双酶切后的pET-22b与IsDNA序列进行重组,得到表达IsDNA的重组表达质粒。
3.一种表达IsDNA序列的工程菌,其特征在于,所述的工程菌含有如权利要求1所述的IsDNA序列或权利要求2所述的重组表达质粒。
4.根据权利要求3所述的工程菌,其特征在于,所述含有IsDNA序列的工程菌为BL21(DE3)/IsDNA,通过如下制备步骤制得:选取大肠杆菌BL21(DE3);利用热击转化方法将重组表达质粒转入大肠杆菌BL21(DE3)中,得到表达IsDNA的工程菌。
5.一种如权利要求4所述的工程菌在生产天然的靛蓝色素中的应用。
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