CN112430617A - 用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒 - Google Patents
用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒 Download PDFInfo
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Abstract
本发明涉及一种用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒。本发明的的质粒上设有蔗糖致死基因sacB,蔗糖致死基因sacB表达分泌型蔗糖果聚糖酶,用于阴性筛选。本发明的方法能在谷氨酸棒杆菌基因组中能够高效快速的实现基因修饰而不带有任何抗性标记。该方法运用基因组双链断裂的反选以及精确的同源重组修复获得目的重组菌株,利用阴性筛选标记sacB能够高效的消除重组菌株中携带的质粒,这克服了稳定性高的过表达质粒难于消除的问题,从而大大提高获得目的重组菌株的效率。该方法快速高效的消除编辑正确的目的菌株中的抗性标记,可以快速实现一个基因修饰。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及一种用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒。
背景技术
谷氨酸棒杆菌(Corynebacterium glutamicum)是从土壤中分离出的非致病菌。该菌在经济上具有重要作用,已被广泛用于工业发酵生产各种氨基酸,如L-谷氨酸和L-赖氨酸。随着直接作用于DNA技术的发展以及谷氨酸棒杆菌全基因组的解析,利用基因工程手段改良谷氨酸棒杆菌成为可能,并逐渐取代经典的随机突变育种。
随着CRISPR基因编辑技术的发展,大大地加速了谷氨酸棒杆菌的育种。然而,在目前建立的编辑系统中大多需要利用双质粒体系,一个用于表达Cas效应蛋白而另一个用于表达crRNA或者修复模板。最终得到的目的菌株是不带质粒的,在编辑成功之后均需要将表达质粒进行消除。在谷氨酸棒杆菌中一个普遍有用的方法是利用温敏型复制子pBLts的过表达质粒,也有文献报道通过将质粒中的per编码的分布蛋白删除使得rep复制子分离不稳定从而利于消除。谷氨酸棒杆菌宿主中,在实验过程中我们发现不知道何种原因质粒中per的删除对于含有rep复制子的质粒的失稳影响并不明显。因此,对于双质粒基因编辑体系,构建第二个兼容的易于消除的过表达质粒将是非常必要的。
发明内容
为解决上述技术问题,本发明构建了新型的带阴性筛选标记的过表达质粒pZF-CrA-HDA,该质粒含有目的基因待编辑处两侧的同源片段以及靶向目的基因的CrRNA,电转到携带效应蛋白Fncpf1的过表达载体的谷氨酸棒杆菌细胞中通过同源重组修复获得目的菌种,将所得的带有双质粒菌株的谷氨酸棒杆菌株培养在无抗的含有10%蔗糖的LBG培养基中能够快速的将质粒消除,从而大大提高获得目的菌种效率。
本发明的第一个目的是提供一种用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒,所述的质粒上设有蔗糖致死基因sacB,所述的蔗糖致死基因sacB表达分泌型蔗糖果聚糖酶,用于阴性筛选。
进一步地,所述的质粒上还设有抗菌素抗性标记。
进一步地,所述的抗菌素抗性标记为氨苄青霉素抗性、卡那霉素抗性、四环素抗性、链霉素抗性、氯霉素抗性中的至少一种。
进一步地,所述的质粒是以pJYS2、pXMJ19、pEC-xk99e或pDXW-8为载体。
本发明的第二个目的是提供所述的用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒的构建方法,包括如下步骤:
S1、以含有蔗糖致死基因sacB的质粒为模板,扩增得到蔗糖致死基因sacB片段;
S2、以含有抗菌素抗性标记的质粒为模板,扩增得到抗菌素抗性标记基因片段;
S3、以载体质粒为模板,扩增得到载体片段;
S4、将蔗糖致死基因sacB片段、抗菌素抗性标记基因片段和载体片段无缝克隆组装得到所述的用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒。
本发明的第三个目的是提供所述的用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒在筛选质粒消除的谷氨酸棒杆菌中的应用。
进一步地,所述的应用是在双质粒基因编辑体系中筛选质粒消除的谷氨酸棒杆菌。
进一步地,所述的应用具体包括如下步骤:
S01、以为所述的用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒为模板,构建表达gRNA的载体;
S02、扩增待编辑的目的基因的上下同源臂片段,连接到S01步骤的表达gRNA的载体;
S03、将温敏型质粒和S02步骤的质粒转化到宿主细胞感受态中对目的基因进行编辑,得到目的菌株;
S04、筛选在抗菌素抗性平板上能够正常生长的菌株,并进行菌落PCR验证,得到正确编辑的菌株;
S05、将正确编辑的菌株在不含有抗菌素,且含有蔗糖的培养基中,在34~36℃培养,去除温敏型质粒和S02步骤的质粒,再去除基因组中的抗菌素抗性基因,筛选在含有蔗糖的平板上生长,且在抗菌素平板上不能生长的菌株。
进一步地,培养基中蔗糖的含量为8~12%。
借由上述方案,本发明至少具有以下优点:
本发明的方法能在谷氨酸棒杆菌基因组中能够高效快速的实现基因修饰而不带有任何抗性标记。该方法运用基因组双链断裂的反选以及精确的同源重组修复获得目的重组菌株,利用阴性筛选标记sacB能够高效的消除重组菌株中携带的质粒,这克服了稳定性高的过表达质粒难于消除的问题,从而大大提高获得目的重组菌株的效率。该方法快速高效的消除编辑正确的目的菌株中的抗性标记,可以快速实现一个基因修饰。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
图1为带有阴性筛选标记的复制型质粒pZF-CrA-HDA的构建过程图,以pZF-CrproB-HD构建过程为例。
图2为对谷氨酸棒杆菌基因组修饰后质粒消除效果图,所用的LBG平板包含10%蔗糖。
具体实施方式
下面结合实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1:带有阴性筛选标记的复制型质粒pZF-CrA-HDA的构建
以质粒pZF-CrproB-HD为例:
带有阴性筛选标记的复制型质粒pZF-CrproB-HD的构建如图1所示。
以质粒pXMJ19为模板,pZF-F1-F和pZF-F1-R为引物,扩增得到片段pZF-F1,为阳性筛选标记片段。以质粒pK18-mobsacB为模板,pZF-F2-F和pZF-F2-R为引物,扩增得到片段pZF-F2,为阴性筛选标记片段。以质粒pJYS2为模板,pZF-F3-F和pZF-F3-R为引物,扩增得到片段pZF-F3,为载体片段。通过无缝克隆试剂盒将pZF-F1、pZF-F2、pZF-F3组装得到的质粒命名为pZF;
以pZF为模板,利用引物CrproB-F/R进行全质粒PCR,引物CrproB-F/R上携带了靶向Corynebacterium glutamicum ATCC13032中proB基因的编码序列,扩增得到得到的产物经过限制性内切酶DpnI消化模板,利用定点突变试剂盒重组得到的质粒将其命名为pZF-CrproB;
以谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032基因组为模板,引物G149K-up-F/R以及G149K-down-F/R扩增得到G149K-up、G149K-down(引物G149K-up-F和G149K-down-R分别携带限制性内切酶Xba I和Xho I的酶切位点);以G149K-up、G149K-down模板,引物G149K-up-F/G149K-down-R扩增得到G149K-up-down。将G149K-up-down和质粒pZF-CrproB用限制性内切酶Xba I和Xho I分别酶切,纯化后将两者酶连,由此得到质粒命名为pZF-CrproB-HD,核苷酸序列如SEQ ID NO.1所示;
构建过程中使用的引物序列如表1,PCR反应条件如表2。
表1.PCR扩增所需引物序列(下划线为酶切位点,加粗为CrRNA靶向序列)
表2.本发明中PCR相关信息
实施例2:获得目的菌株的方法
以获得pck基因敲除和lyC基因过表达的菌株为例:
将能够表达效应蛋白Fncpf1的温敏型质粒pJYS1转化到Corynebacteriumglutamicum ATCC13032感受态细胞中。在卡那霉素抗性平板筛选能正常生长的菌株Corynebacterium glutamicum ATCC13032/pJYS1。将pZF-CrproB-HD转化到所得菌株Corynebacterium glutamicum ATCC13032/pJYS1制备成的感受态细胞中。利用验证引物G149K-F/R对在卡那霉素和氯霉素双抗平板生长的菌株进行菌落PCR验证,筛选能够扩增出1000bp左右目的条带的菌株。所获得的正确编辑的菌株培养在无抗的含有10%蔗糖的LBG液体小瓶中培养24h,稀释涂布在含有10%蔗糖LBG平板中,通过筛选在含有10%蔗糖LBG平板中能够生长,添加双抗的平板不能生长的菌株,所得菌株即为质粒消除菌株。
将质粒消除菌株进行测序,测序结果正确的即为基因组突变成功菌株。
以上所述方法不仅适用于谷氨酸棒杆菌,对其它任何对蔗糖和卡那霉素、氯霉素抗性敏感的革兰氏阴性或阳性菌株都适用,因此这些内容都属于本发明的保护范围。
以上仅是本发明的优选实施方式,并不用于限制本发明,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和变型,这些改进和变型也应视为本发明的保护范围。
序列表
<110> 江南大学
<120> 用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒
<160> 15
<170> PatentIn version 3.3
<210> 1
<211> 8259
<212> DNA
<213> (人工序列)
<400> 1
tctacgtgta tacgatggta acgctatgaa tgatacgcag aacacacctg aaagcgttcg 60
attacgggat aatctcccaa cgccaaccca aatggcgccg gtgacagggc ttcctgtcac 120
cccctacagc caggaagcaa gcatcggtgc gagcttcccg gcagtggatc cggacaccaa 180
agacagcgcc gcatacggac atgaatccgg aatgcgtgag cgcatctcca acgctaagcg 240
agtggtggtg aaaattggtt cgtcctcatt gactaacgat gaggacggac acaccgtcga 300
tcccaaccgc atcaacacta ttgtcaatgc cttgcaagca cgcatggaag ctggctcgga 360
cctcatcgtt gtgtcctctg gcgcagtggc cgcgggaatg gccccgcttg gattgagcac 420
ccggcccacg gaattggcag tcaagcaggc tgcagcagca gtggggcaag ttcacctcat 480
gcaccagtgg ggacgttctt ttgcccggta tggtcgcccc atcggccagg tgcttcttac 540
cgcagctgat gcaggaaagc gtgatcgtgc gaggaatgcg cagcgtacca tcgacaagct 600
gcgcattttg ggcgcggttc ctatcgtcaa tgaaaatgac accgtggcta ctactaaggt 660
gaattttggt gacaacgacc gacttgctgc aattgtggcg cacctggtgt cggctgatgc 720
tttggtgctg ctcagtgacg tggatggact ttttgataaa aaccctactg atcccaccgc 780
gaagtttatt tccgaggttc gtgacggcaa tgatttgaaa ggtgtcattg ccggcgacgg 840
cggaaaagtg ggcaccggtg gcatggcatc aaaggtgtct gctgcacgtt tggcttcccg 900
aagtggcgtg cctgtgctgt tgacctctgc ggcaaacatt ggcccagcac tggaagacgc 960
ccaggtgggc actgtattcc accccaagga caaccgcctc tccgcgtgga agttctgggc 1020
tttgtatgcc gcagatactg caggaaagat ccgactcgat gacggcgcgg tggaagcagt 1080
gacctccggt ggtaaatctt tgctggctgt gggcattact gaaatcattg gtgatttcca 1140
gcagggtgag atcgtggaga tcttgggacc tgccggccaa atcatcgggc gaggcgaggt 1200
gtcctacgat tctgatacct tgcaatcaat ggttggtatg caaacgcagg accttccaga 1260
tggcatgcag cgcccggtag tgcatgcaga ttatctgtcc aactacgcca gccgcgcgta 1320
aagcgcgggc ctgctggtgg cgggtggcgt cgaaaagcat ttttaaagga gtttaagacg 1380
atgaagtttg ttatgtatcc gcatttgtgg gagtccacga ccgctgtcat tgagggtggc 1440
ggacatgagc gggttgagga tattaaagat gcagacttca ttttctttaa tggttcagcg 1500
ccggagttcc cggatttgcc ggagaacatc aagttcgtgc aggcctccat ggcgggtatt 1560
gatgcgctgg tcaagcgtgg tgtcgtcaat gagaaggcac gttggctcga ggagattttt 1620
gagggggagg gagtcgagga agagccagag cagaaggcgg gggaaccgtt ctctgccgac 1680
agcgtgagcc cccttaaaaa tcaggccggg gaggaaccgg ggagggatca gagctaggag 1740
cgagacaccc taaagggggg gaaccgtttt ctgctgacgg tgtttcgttt attagttttc 1800
agcccgtgga tagcggaggg tgagggcaag tgagagccag agcaaggacg ggacccctaa 1860
aggggggaac cgttttctgc tgacggtgtt tcgtttatta gttttcagcc cgtggacggc 1920
cgcgtttagc ttccattcca agtgcctttc tgacttgttg gatgcgcctt tcactgacac 1980
ctagttcgcc tgcaagctca cgagtcgagg gatcagcaac cgattgagaa cgggcatcca 2040
ggatcgcagt tttgacgcga agttcgagca actcgcctgt catttctcgg cgtttgtttg 2100
cttccgctaa tcgctgtcgc gtctcctgcg catacttact ttctgggtca gcccatctgc 2160
gtgcattcga tgtagctgcg ccccgtcgcc ccatcgtcgc tagagctttc cgccctcggc 2220
tgctctgcgt ttccacccga cgagcaggga cgactggctg gcctttagcc acgtagccgc 2280
gcacacgacg cgccatcgtc aggcgatcac gcatggcggg aagatccggc tcccggccgt 2340
ctgcaccgac cgcctgggca acgttgtacg ccacttcata cgcgtcgatg atcttggcat 2400
cttttaggcg ctcaccagca gctttgagct ggtatcccac ggtcaacgcg tggcgaaacg 2460
cggtctcgtc gcgcgctgcg cgctctggat ttgtccagag cactcgcacg ccgtcgatca 2520
ggtcgccgga cgcgtccagg gcgctcggca ggctcgcgtc caaaatcgct agcgccttgg 2580
cttctgcggt ggcgcgttgt gccgcttcaa tgcgggcgcg tccgctggaa aagtcctgct 2640
caatgtactt tttcggcttc tgtgatccgg tcatcgttcg agcaatctcc attaggtcgg 2700
ccagccgatc cacacgatca tgctggcagt gccatttata ggctgtcgga tcgtctgaga 2760
cgtgcagcgg ccaccggctc agcctatgcg aaaaagcctg gtcagcgccg aaaacacgag 2820
tcatttcttc cgtcgttgca gccagcaggc gcatatttgg gctggtttta cctgctgcgg 2880
catacaccgg gtcaatgagc cagatgagct ggcatttccc gctcagcgga ttcacgccga 2940
tccaagccgg cgctttttct aggcgtgccc atttctctaa aatcgcgtag acctgcgggt 3000
ttacgtgctc aatcttcccg ccggcctggt ggctgggcac atcgatgtca agcacgatca 3060
ccgcggcatg ttgcgcgtgc gtcagcgcaa cgtactggca ccgcgtcagc gcttttgagc 3120
cagcccggta gagctttggt tgggtttcgc cggtatccgg gtttttaatc caggcgctcg 3180
cgaaatctct tgtcttgctg ccctggaagc tttcgcgtcc caggtgagcg agcagttcgc 3240
ggcgatcttc tgccgtccag ccgcgtgagc gcagcgctag ttcggggtgg gtgtcgaaca 3300
gatcggcgga aaatttccac gcgctagctg tgactgtgtc ctgcggatcg gctagagtca 3360
tgtcttgagt gctttctccc agctgatgac tgggggttag ccgacgccct gtgagttccc 3420
gctcacgggg cgttcaactt tttcaggtat ttgtgcagct tatcgtgttt tcttcgtaaa 3480
tgaacgctta actaccttgt taaacgtggc aaataggcag gattgatggc tttcatagaa 3540
ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt cggtcatttc 3600
gaagggcacc aataactgcc ttaaaaaaag atctgtacat cgatatcata tgcaagggca 3660
catatacctg ccgttcacta ttatttagtg aaatgagata ttatgatatt ttctgaattg 3720
tgattaaaaa ggcaacttta tgcccatgca acagaaacta taaaaaatac agagaatgaa 3780
aagaaacaga tagatttttt agttctttag gcccgtagtc tgcaaatcct tttatgattt 3840
tctatcaaac aaaagaggaa aatagaccag ttgcaatcca aacgagagtc taatagaatg 3900
aggtcgaaaa gtaaatcgcg cgggtttgtt actgataaag caggcaagac ctaaaatgtg 3960
taaagggcaa agtgtatact ttggcgtcac cccttacata ttttaggtct ttttttattg 4020
tgcgtaacta acttgccatc ttcaaacagg agggctggaa gaagcagacc gctaacacag 4080
tacataaaaa aggagacatg aacgatgaac atcaaaaagt ttgcaaaaca agcaacagta 4140
ttaaccttta ctaccgcact gctggcagga ggcgcaactc aagcgtttgc gaaagaaacg 4200
aaccaaaagc catataagga aacatacggc atttcccata ttacacgcca tgatatgctg 4260
caaatccctg aacagcaaaa aaatgaaaaa tatcaagttt ctgaatttga ttcgtccaca 4320
attaaaaata tctcttctgc aaaaggcctg gacgtttggg acagctggcc attacaaaac 4380
gctgacggca ctgtcgcaaa ctatcacggc taccacatcg tctttgcatt agccggagat 4440
cctaaaaatg cggatgacac atcgatttac atgttctatc aaaaagtcgg cgaaacttct 4500
attgacagct ggaaaaacgc tggccgcgtc tttaaagaca gcgacaaatt cgatgcaaat 4560
gattctatcc taaaagacca aacacaagaa tggtcaggtt cagccacatt tacatctgac 4620
ggaaaaatcc gtttattcta cactgatttc tccggtaaac attacggcaa acaaacactg 4680
acaactgcac aagttaacgt atcagcatca gacagctctt tgaacatcaa cggtgtagag 4740
gattataaat caatctttga cggtgacgga aaaacgtatc aaaatgtaca gcagttcatc 4800
gatgaaggca actacagctc aggcgacaac catacgctga gagatcctca ctacgtagaa 4860
gataaaggcc acaaatactt agtatttgaa gcaaacactg gaactgaaga tggctaccaa 4920
ggcgaagaat ctttatttaa caaagcatac tatggcaaaa gcacatcatt cttccgtcaa 4980
gaaagtcaaa aacttctgca aagcgataaa aaacgcacgg ctgagttagc aaacggcgct 5040
ctcggtatga ttgagctaaa cgatgattac acactgaaaa aagtgatgaa accgctgatt 5100
gcatctaaca cagtaacaga tgaaattgaa cgcgcgaacg tctttaaaat gaacggcaaa 5160
tggtacctgt tcactgactc ccgcggatca aaaatgacga ttgacggcat tacgtctaac 5220
gatatttaca tgcttggtta tgtttctaat tctttaactg gcccatacaa gccgctgaac 5280
aaaactggcc ttgtgttaaa aatggatctt gatcctaacg atgtaacctt tacttactca 5340
cacttcgctg tacctcaagc gaaaggaaac aatgtcgtga ttacaagcta tatgacaaac 5400
agaggattct acgcagacaa acaatcaacg tttgcgccga gcttcctgct gaacatcaaa 5460
ggcaagaaaa catctgttgt caaagacagc atccttgaac aaggacaatt aacagttaac 5520
aaataaaaac gcaaaagaaa atgccgatgg gtaccgagcg aaatgaccga ccaagcgacg 5580
cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc 5640
ggagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgccggg 5700
gatcagcccc ggatgctttg gatacggtct atgagctggc agcgtatttg accgatccgg 5760
acacctggga taatgtgtgg attttgtcgg atcagcttga gtaggacaaa tccgccgagc 5820
ttcgacgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac 5880
caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc 5940
tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa 6000
gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc 6060
tcatccggag ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca 6120
cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata 6180
ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga 6240
aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc 6300
ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc 6360
cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat 6420
tcaggttcat catgccgtct gtgatggctt ccatgtcggc agaatgctta atgaattaca 6480
acagtactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc 6540
ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc 6600
gccttctatg aaaggttggg cttcggaatc gttttccggg acgtcatgac caaaatccct 6660
taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 6720
tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 6780
gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 6840
agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 6900
aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 6960
gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 7020
gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 7080
tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 7140
agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 7200
cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 7260
gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 7320
gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 7380
ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 7440
cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 7500
cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatgaag cttaagagtt 7560
tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttaatttg atgcctggca 7620
gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcgcaac gttcaaatcc 7680
gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca gataaaacga 7740
aaggcccagt ctttcgactg agcctttcgt tttattggaa ttccgtcaat gaaaatgaca 7800
ccgtggatct acaacagtag aaattcggat ccattatacc taggactgag ctagctgtca 7860
agctctagac agggcggatt catcatcacc ggcgaaaagc cagagcgctg gattttgcag 7920
accgactttg aaaacgacga agcagttggc tacctggctg accgtctggc caagttgggc 7980
attgaggacg ggcttcgtaa ggcaggagca catgtgggtg caaacgtcac catcggaggc 8040
atttccttcg agtgggagcc aatgaccacc gctggcgacg atccagtcct taccggacgt 8100
ggcaccgatg tgcgccttga acagacctct cgtatctctg ctgcagagcg taaacgcgca 8160
tctcaggtac gtcgtggcct catcgatgag ttggattatg gcgaggacca agaggcttcc 8220
cgcgaacgct gggaaggata aaaccgagca cttttcagg 8259
<210> 2
<211> 24
<212> DNA
<213> (人工序列)
<400> 2
cttcggaggg caatcagctg ttgc 24
<210> 3
<211> 36
<212> DNA
<213> (人工序列)
<400> 3
aagggatttt ggtcatgacg tcccggaaaa cgattc 36
<210> 4
<211> 41
<212> DNA
<213> (人工序列)
<400> 4
atcgatatca tatgcaaggg cacatatacc tgccgttcac t 41
<210> 5
<211> 32
<212> DNA
<213> (人工序列)
<400> 5
agctgattgc cctccgaagc ccaacctttc at 32
<210> 6
<211> 22
<212> DNA
<213> (人工序列)
<400> 6
tcatgaccaa aatcccttaa cg 22
<210> 7
<211> 22
<212> DNA
<213> (人工序列)
<400> 7
cccttgcata tgatatcgat gt 22
<210> 8
<211> 44
<212> DNA
<213> (人工序列)
<400> 8
cgtcaatgaa aatgacaccg tggatctaca acagtagaaa ttcg 44
<210> 9
<211> 43
<212> DNA
<213> (人工序列)
<400> 9
ccacggtgtc attttcattg acggaattcc aataaaacga aag 43
<210> 10
<211> 25
<212> DNA
<213> (人工序列)
<400> 10
gctctagaca gggcggattc atcat 25
<210> 11
<211> 48
<212> DNA
<213> (人工序列)
<400> 11
attcacctta gtagtagcca cggtgtcatt ttcattgacg ataggaac 48
<210> 12
<211> 40
<212> DNA
<213> (人工序列)
<400> 12
caccgtggct actactaagg tgaattttgg tgacaacgac 40
<210> 13
<211> 25
<212> DNA
<213> (人工序列)
<400> 13
ccctcgagcc aacgtgcctt ctcat 25
<210> 14
<211> 19
<212> DNA
<213> (人工序列)
<400> 14
caccgtggct actactaag 19
<210> 15
<211> 18
<212> DNA
<213> (人工序列)
<400> 15
cttagccaaa cgagtcgt 18
Claims (9)
1.一种用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒,其特征在于,所述的质粒上设有蔗糖致死基因sacB,所述的蔗糖致死基因sacB表达分泌型蔗糖果聚糖酶,用于阴性筛选。
2.根据权利要求1所述的用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒,其特征在于,所述的质粒上还设有抗菌素抗性标记。
3.根据权利要求2所述的用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒,其特征在于,所述的抗菌素抗性标记为氨苄青霉素抗性、卡那霉素抗性、四环素抗性、链霉素抗性、氯霉素抗性中的至少一种。
4.根据权利要求1所述的用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒,其特征在于,所述的质粒是以pJYS2、pXMJ19、pEC-xk99e或pDXW-8为载体。
5.一种权利要求1-4任一项所述的用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒的构建方法,其特征在于,包括如下步骤:
S1、以含有蔗糖致死基因sacB的质粒为模板,扩增得到蔗糖致死基因sacB片段;
S2、以含有抗菌素抗性标记的质粒为模板,扩增得到抗菌素抗性标记基因片段;
S3、以载体质粒为模板,扩增得到载体片段;
S4、将蔗糖致死基因sacB片段、抗菌素抗性标记基因片段和载体片段无缝克隆组装得到所述的用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒。
6.权利要求1-4任一项所述的用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒在筛选质粒消除的谷氨酸棒杆菌中的应用。
7.根据权利要求6所述的应用,其特征在于,所述的应用是在双质粒基因编辑体系中筛选质粒消除的谷氨酸棒杆菌。
8.根据权利要求6所述的应用,其特征在于,所述的应用具体包括如下步骤:
S01、以为所述的用于谷氨酸棒杆菌基因改造的带有阴性筛选标记的质粒为模板,构建表达gRNA的载体;
S02、扩增待编辑的目的基因的上下同源臂片段,连接到S01步骤的表达gRNA的载体;
S03、将温敏型质粒和S02步骤的质粒转化到宿主细胞感受态中对目的基因进行编辑,得到目的菌株;
S04、筛选在抗菌素抗性平板上能够正常生长的菌株,并进行菌落PCR验证,得到正确编辑的菌株;
S05、将正确编辑的菌株在不含有抗菌素,且含有蔗糖的培养基中,在34~36℃培养,去除温敏型质粒和S02步骤的质粒,再去除基因组中的抗菌素抗性基因,筛选在含有蔗糖的平板上生长,且在抗菌素平板上不能生长的菌株。
9.根据权利要求8所述的应用,其特征在于,培养基中蔗糖的含量为8~12%。
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CN114480467A (zh) * | 2022-02-24 | 2022-05-13 | 江南大学 | 一种在棒状杆菌中辅助sacB基因编辑系统的CRISPR-cpf1筛选工具 |
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CN113652442A (zh) * | 2021-08-27 | 2021-11-16 | 江南大学 | 一种棒状杆菌质粒复制子的改造方法及其产品 |
CN114480467A (zh) * | 2022-02-24 | 2022-05-13 | 江南大学 | 一种在棒状杆菌中辅助sacB基因编辑系统的CRISPR-cpf1筛选工具 |
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