新型Red重组质粒pRO38的构建及Red系统的定向进化

[目的]构建新型Red同源重组质粒,对red基因进行分子定向进化,获取高重组效率的red突变基因。[方法]采用PCR的方法扩增不同的功能片断,通过体外同源重组的方法构建Red同源重组质粒pRO38。采用双链重组的实验验证质粒的同源重组功能后,随机突变red基因,通过修复neo缺失突变的方法筛选具有高重组效率的red基因突变质粒库。[结果]构建了长为5 593 bp的pRO38质粒,pRO38质粒具备正常的重组功能,可以有效地将kan-sac B片段插入大肠杆菌染色体。对pRO38中的red基因进行随机突变后,获得了约60个成功修复了neo缺失突变的菌落,而原始的pRO38质粒则无菌落生长。[结论]构建了新型的Red同源重组质粒。通过定向进化的手段,成功获得了高重组效率的red突变基因库,为从根本上提高Red重组效率打下了基础。     

一种快速、精确构建大肠杆菌组氨酸营养缺陷型的方法*

将表达Red体内重组蛋白的质粒pKD46转化大肠杆菌DH5α,用 5′端与组氨酸基因同源 ,3′端与卡那霉素抗性基因同源的引物获得具有卡那霉素抗性基因的PCR产物 ,然后电击转化DH5α,在λRed重组系统的帮助下 ,通过卡那霉素抗性基因两侧的组氨酸基因序列在体内与大肠杆菌染色体上的组氨酸基因发生同源重组 ,置换了DH5α组氨酸操纵元中的hisDCB基因 ,最后利用卡那霉素抗性基因两端的FRT位点 ,通过FTP位点专一性重组将卡那霉素抗性基因去除 ,最终获得了不具抗性的大肠杆菌组氨酸营养缺陷型菌株。为在大     

一种快速、精确构建大肠杆菌组氨酸营养缺陷型的方法

将表达Red体内重组蛋白的质粒pKD46转化大肠杆菌：DH5α,用5′端与组氨酸基因同源,3′端与卡那霉素抗性基因同源的引物获得具有卡那霉素抗性基因的PCR产物,然后电击转化DH5α,在λRed重组系统的帮助下,通过卡那霉素抗性基因两侧的组氨酸基因序列在体内与大肠杆菌染色体上的组氨酸基因发生同源重组,置换了DH5α组氨酸操纵元中的hisDCB基因,最后利用卡那霉素抗性基因两端的FRT位点,通过FTP位点专一性重组将卡那霉素抗性基因去除,最终获得了不具抗性的大肠杆菌组氨酸营养缺陷型菌株。为在大肠杆菌及其他菌株中快速、精确的构建营养缺陷型菌株提供了有益的参考。     

肺炎支原体铁吸收调节蛋白基因的克隆表达与纯化

[目的]克隆肺炎支原体铁吸收调节蛋白(Fur)基因并纯化Fur蛋白,为研究其生物学功能奠定基础。[方法]利用Clustal Omega分析肺炎支原体Fur蛋白及其同源序列并用MEGA6.0构建进化树,通过PCR扩增Fur基因并对其进行双酶切,然后连接到p ET28a,得到重组载体p ET28a-Fur,将其转化大肠杆菌BL21(DE3),利用IPTG诱导Fur蛋白表达,并通过亲和层析纯化Fur蛋白。[结果]多序列比对和进化树分析表明Mp含有一个Fur蛋白。克隆得到大小为477 bp的Fur基因,编码159个氨基酸。得到重组表达质粒p ET28a-Fur,该质粒能在大肠杆菌中能高效表达,并纯化得到重组Fur蛋白。[结论]成功克隆获得Mp Fur基因,在大肠杆菌中高效表达并获得高纯度Fur蛋白。     

利用Red系统快速敲除家蚕核型多角体病毒orf60基因

用Red重组系统和最近构建的家蚕核型多角体病毒(BmNPV)bacmid在大肠杆菌BW25113中快速地敲除BmNPVorf60基因。从大肠杆菌BmDH10Bac中提取BmNPVbacmid,将其电转化到含有质粒pKD46(能表达Red重组酶)的大肠杆菌菌株BW25113中,获得了可用于BmNPV基因打靶的菌株BW25113-Bac。设计一对长63bp的引物(5′端为orf60基因的左右同源臂,长45bp;3′端长18bp,为氯霉素抗性基因(cat)的首尾序列),以pKD3质粒(含cat)为模板,PCR扩增携带orf60左右同源臂的cat,即打靶线性化片段。将该线性化片段电转入BW25113-Bac菌株,在Red重组酶的作用下,线性化片段与BmNPVbacmid中的orf60基因发生同源重组。设计3对特异引物,用PCR方法证明cat成功地替换了BmNPVorf60基因。重组bacmid DNA转染BmN细胞后,Western blot分析未检测到orf60基因的表达。     

利用Red系统快速改构含鼠β-酪蛋白基因的细菌人工染色体

采用Red系统介导的同源重组方法对含有鼠β-酪蛋白基因的RPCI23-440C1BAC进行快速改构。首先通过PCR方法,获得两端带有鼠β-酪蛋白基因同源序列的tPAm-Zeo同源重组片段,然后将此同源重组片段电击转化至已含有编码Red重组酶质粒的RPCI23-440C1BAC菌中,在λRed重组系统的帮助下,通过同源重组片段两端与RPCI23-440C1中β-酪蛋白同源的序列在菌体内与β-酪蛋白基因发生同源重组,将其置换。最后利用Zeocin抗性基因两侧的FRT位点,通过FLP位点专一性重组将抗性基因剔除。经Southern blot和序列分析鉴定表明,获得了重组正确且无编码两种重组酶质粒的tPAm-RPCI23-440C1BAC克隆。     

细菌内同源重组法制备FMDV聚蛋白编码基因重组腺病毒

采用PCR方法从重组质粒pMD18_T/PP中扩增出FMDV的聚蛋白(PP)编码基因,再亚克隆至腺病毒穿梭质粒中,形成重组穿梭质粒rpAd_CMV/PP;将获得的重组穿梭质粒与腺病毒骨架载体通过在大肠杆菌内质粒间同源重组获得重组腺病毒质粒rpAd/PP。将腺病毒载体线性化后用脂质体介导转染293细胞从而获得含有口蹄疫病毒PP编码基因的重组腺病毒。通过倒置显微镜观测,可见明显的细胞病变,利用荧光显微镜可观测到报告基因绿色荧光蛋白的表达,并在电镜下观察到FMDV的空衣壳。结果证明已成功获得了含有口蹄疫病毒PP编码基因的重组腺病毒rAd/PP,并成功表达组装FMDV空衣壳,为FMDV腺病毒活载体疫苗的研究奠定了基础。     

猪繁殖与呼吸综合征病毒M+N基因重组腺病毒载体的构建

本研究构建了M+N基因的重组腺病毒载体。首先用反转录聚合酶链反应(RT-PCR)的方法分别扩增出猪繁殖与呼吸综合征病毒M基因和N基因,将两者用口蹄疫病毒2A序列串联起来,将连接好的M+N基因插入腺病毒穿梭载体pAdTrack-CMV,经筛选获得了重组质粒pAdTrack-CMV/M+N;然后在大肠杆菌BJ5183内将此重组质粒和腺病毒骨架载体pAdEasy-1进行同源重组,获得了插有外源基因的重组腺病毒质粒pAd/M+N;最后,经PacⅠ酶切线性化后转染HEK-293细胞,成功获得含有M+N基因的重组腺病毒,为重组腺病毒活载体疫苗的研究奠定基础。     

谷氨酸棒状杆菌CRISPR-Cpf1和Cre/loxP基因敲除技术的比较

【背景】谷氨酸棒状杆菌的基因敲除系统较为匮乏且效率不高,难以对其进行代谢工程改造,不利于高性能工业菌株的构建及规模生产。【目的】分别采用CRISPR-Cpf1和Cre/loxP基因敲除系统对谷氨酸棒状杆菌ATCC13032(CorynebacteriumglutamicumATCC13032)基因组上的argR和argF基因进行敲除,比较两种敲除方法的优缺点,为合理选择敲除系统提供依据。【方法】特异性重组的Cre/loxP敲除系统是首先利用同源重组将基因组上的靶基因替换为两端带有重组位点loxP的kanR片段,然后由重组酶Cre识别loxP位点并发生重组反应,从而去除替换到基因组上的kanR片段,进一步利用质粒的温敏特性将其消除,从而实现靶基因的敲除。CRISPR-Cpf1敲除系统是利用Cpf1对pre-crRNA进行加工,形成的成熟crRNA引导Cpf1识别和结合到靶DNA的特定序列上并切割双链DNA分子,通过同源重组作用去除靶基因,基于质粒自身的温敏特性将其消除,从而完成基因敲除的整个过程。【结果】Cre/lox P系统可在8N+2 d内完成N轮迭代基因敲除,而CRISPR-Cpf1系统可在5N+2d内完成N轮迭代基因无痕敲除,理论上还可以一次对多个靶位点进行编辑,效率更高,但存在同源重组效率较低、假阳性率高等缺点。【结论】与Cre/loxP系统相比,CRISPR-Cpf1辅助的同源重组基因敲除方法可省时、省力地实现基因的无痕敲除,理论上还可实现多个基因的同时敲除、总体效率更高,然而编辑效率还有提高的空间。     

利用λ-Red重组系统和平衡致死系统改造抗药性致腹泻工程疫苗

质粒pMM085是含有猪毒素源性大肠杆菌(ETEC)的黏附素K88与无毒肠毒素LTA-B 基因的重组质粒,含氯霉素抗性基因,由此构建的菌苗株带有抗药性。利用平衡致死系统改建此疫苗株,即将质粒上的氯霉素抗性基因cat替换成asd基因,并把新构建的质粒转移到缺失asd基因的大肠杆菌X6097中。但由于质粒pMM085是一个23kD的大质粒,传统的基因工程操作不易进行,利用λ-Red重组系统,将表达Red重组蛋白的质粒pKD46转化含pMM085的大肠杆菌X6097,并用两端各带有39ntcat基因同源区、含全长asd基因的PCR产物电击转化此感受态细胞,在λ-Red重组系统的帮助下,成功实现了asd基因对cat基因的置换。     

铜绿假单胞菌中快速基因操作工具的开发和应用

针对基因的操作,包括缺失和插入基因、替换基因元件(如启动子)、融合荧光蛋白基因、构建原位的基因报告系统,是大多数生物技术实验室的必备技术。目前广泛使用的基于2次同源重组的基因操作方法,在构建质粒、转化和筛选方面较为繁琐。另外使用该方法进行长片段敲除的效率较低。为了简化基因操作的流程,本研究构建了一个最小化的铜绿假单胞菌(Pseudomonas aeruginosa)整合型质粒pln2,只需将目标基因内部一段序列克隆到pln2质粒并导入细菌,由于质粒不能在细菌内自我复制而只能通过单次等位基因交换整合到基因组上,从而使目的基因断裂为两部分而失去活性。在pln2的基础上开发了一整套工具质粒适用于基因组的不同操作,包括融合荧光蛋白基因、替换基因元件(如启动子)、构建原位的转录型荧光报告系统。此外,本研究借助该系统成功实现超长片段基因簇的敲除,单次可以敲除长达270 kb的片段。     

Marker rescue from the Nicotiana tabacum plastid genome using a plastid/Escherichia coli shuttle vector

We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICEI sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the CoIE1 ori for maintenance in E. coli and a spectinomcyin resistance gene (aadA) for selection in both systems. In addition, pNICERl carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICERl was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE 1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology.     

新型酵母蛋白表位标记和基因敲除质粒系统的构建及可行性验证

【目的】构建一套用于酿酒酵母基因功能研究的质粒。该套质粒结合pUG系列和pFA6a系列的优点,同时采用同尾酶实现蛋白表位标签的串联插入。【方法】利用PCR技术分别克隆pUG系列质粒的lox P位点、pFA6a质粒多酶切位点和ADH1终止子模块;通过重组连接各片段,构建pCLHN-TRP和pCLHN-URA质粒。在此基础上利用同尾酶实现多种蛋白表位标签的单个或串联重复插入,获得一系列蛋白表位标记质粒。最后,以ATG1、COX4和NHX1为例验证本质粒系列的性能。【结果】在本项工作中,我们共构建2种基因敲除用质粒和17种表位标记用质粒(涵盖1-8 FLAG、1-12 V5、3-9 HA、2-8MYC、GFP和m Cherry)。在几个靶基因上的应用证实了本套质粒的实用性。尤其值得指出的是,通过组合采用不同重复度的串联表位标签,在同一张膜上同时检测表达差异极大的不同蛋白而不使高表达蛋白信号饱和成为可能。【结论】本文所构建的pCLHN质粒系列是对现有酵母质粒工具的有益补充。     

Mapping of the sor genes for l-sorbose degradation in the chromosome of Klebsiella pneumoniae

Summary A series of mutants was isolated in Klebsiella pneumoniae strain 1033, among them mutants unable to grown on l-sorbose. Different R' plasmids carrying the sor genes and other surrounding chromosomal genes were also isolated. Each plasmid contained the structural genes sorA for an Enzyme II of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system, sorD for a d-glucitol 6-phosphate dehydrogenase, sorE for an l-sorbose 1-phosphate reductase, and the corresponding regulator gene sorR. These structural genes are coordinately expressed and inducible by l-sorbose. Cis-dominant and pleiotropic mutations rendering the expression of the sor genes constitutive or eliminating it were isolated. Complementation of a series of mutations in Escherichia coli K12 and K. pneumoniae by various R' and F' plasmids and by P1 transduction in K. pneumoniae located the sor genes within the following gene sequence: rbs rha pfkA metB ppc argH ilv btuB rpoB metA ace sor pgi malB uvrA. The rbs-ilv gene loci tightly linked in E. coli K12 at 84 min, are separated in the map of K. pneumoniae 1033 and located at 86 and 89 min, respectively.     

The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli

The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.     

谷氨酸棒杆菌中基于CRISPR/Cas9的多位点碱基编辑系统的优化

作为新型的基因组编辑工具,碱基编辑技术结合了CRISPR/Cas系统的定位功能和碱基脱氨酶的编辑功能,可实现特定位点的碱基突变,具有不产生双链DNA断裂,无需外源模板且不依赖染色体DNA同源重组的优势.目前,研究者们已在重要的工业生产菌株谷氨酸棒杆菌(Corynebacterium glutamicum)中开发了多种碱...     

微嗜酸寡养单胞菌中的漆酶对黄曲霉毒素B1降解脱毒的生物活性

[目的]探究微嗜酸寡养单胞菌中的漆酶对AFB1的降解活性,并确定漆酶在菌株CW117降解代谢AFB1过程中的贡献.[方法]从微嗜酸寡养单胞菌基因组中,共筛选到两个漆酶基因lc1和lc2,并用大肠杆菌BL21外源表达蛋白rLC1和rLC2,在体外检测其对AFB1的降解活性.同时参考前人报道,研究了氧化性辅剂对漆酶AFB1...     

Escherichia coli mutants incapable of supporting replication of F-like plasmids at high temperature: isolation and characterization of mafA and mafB mutants.

Mutants of Escherichia coli K-12 defective in replication of F-like plasmids at a high temperature (42 degrees C) were found among threonine-independent (Thr+) revertants of a threonine-requiring F' stain after localized mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Transduction experiments with phage P1 permitted us to divide these mutations into two classes with respect to man location; some mutations were located between thr and ara at about 0.8 min, very close to maf-1 reported previously (Wada et al., J. Mol. Biol. 108:25-41, 1976 and the others probably were located between leu and azi at about 1.8 min. The former class of mutants designated mafA exhibited the same plasmid specificity as maf-1; replication of plasmids F and ColVB trp, but not R386 or R222, were affected at a high temperature. By contrast, the latter mutants designated mafB were defective in replication of nay of these plasmids at a high temperature. When a culture of mafA mutants carrying an F' plasmid was transferred from 30 to 42 degrees C, the plasmid replication as determined by incorporation of [3H]thymidine into covalently closed circular F DNA was markedly inhibited. Under certain conditions, the temperature shift-up caused severe growth inhibition of the mutant cells. Examination of merodiploids (mafA/FmafA+) for plasmid maintenance suggested that the two mafA mutations tested (mafA23 and mafA36) were both dominant, at least partially, over the wild-type mafA+ allele. These properties of the mafA mutants, manifested at the restrictive temperature, are similar to those previously reported for the maf-1 mutant. Taken together with other evidence it is likely that these mutations affect either the same gene (mafA) or a set of closely linked genes, playing a specific role in autonomous plasmid replication in E. coli.