基于原生质体法保加利亚乳杆菌电转化条件的研究

目的:研究不同因素对保加利亚乳杆菌原生质体法电转化效率的影响,通过研究电转化的最适条件,以提高电转化的效率。方法:以保加利亚乳杆菌为受体菌株,大肠杆菌-乳酸菌穿梭型质粒pMG36e为载体,通过原生质体电转化的方法研究溶菌酶浓度、质粒浓度、电转化参数、复苏培养基组成对电转化效率的影响。结果:用SMM在37℃下制备溶菌酶(30 mg/mL)对细胞进行酶解,直至通过显微镜发现约60%的原生质体形成。用1μL质量浓度为1.2μg/μL的质粒pMG36e,在电场强度7.5 kV/cm,电阻200Ω,电容25μF的条件进行电击转化,以含有0.5 mol/L蔗糖和20 mmol/L的MgCl_2、CaCl_2且无吐温80的MRS再生培养基复苏培养2.5 h,并以含红霉素的MRS平板筛选,获得1.42×10~5CFU/μgDNA的电转化效率。结论:本研究实现了保加利亚乳杆菌的高效遗传转化,并为遗传育种提供了技术支持。     

鼠李糖乳杆菌电转化条件的研究

目的:研究不同因素对鼠李糖乳杆菌电转化效率的影响,探讨电转化的最适条件,提高电转化效率。方法:以鼠李糖乳杆菌05-28为受体菌株,大肠杆菌-乳酸菌穿梭型质粒p MG36e为载体,通过原生质体电转化的方法研究了质粒浓度、电击缓冲液、电转化参数、复苏培养基组成及复苏培养时间对电转化效率的影响。结果:利用1μL质量浓度为1μg/μL的质粒p MG36e,在电场强度8 k V/cm,电阻200Ω,电容25μF的条件下进行电击转化,以含有0.5 mol/L蔗糖和20 mmol/L的MgCl_2、CaCl_2且无吐温80的MRS再生培养基复苏培养2.5 h,并采用添加红霉素的筛选培养基,获得1.66×105CFU/μg DNA的电转化效率。结论 :实现了鼠李糖乳杆菌的高效遗传转化,为遗传育种提供了技术支持。     

明串珠菌电转化的优化

为了构建食品级基因工程受体菌,课题对明串珠菌的电转化条件进行了优化.以大肠杆菌-明串珠菌穿梭质粒pCW4为载体,以明串珠菌为受体,文章优化了细胞壁处理方式、电击参数、复苏时间等与电转化相关的因素.结果表明:用氨苄浓度0.7μg/mL的MRS培养基培养明串珠菌至OD600为0.5左右,以PBS作缓冲液,收获制成感受态细胞,与1μg DNA混合,在电场强度12 kV/cm、电阻200 Ω、电容25μF下电击转化,以含80 mmol/L MgCl2的MRS培养基复苏培养3h,得到最高的转化效率为1.75×105 cfu/μg DNA.     

嗜碱芽孢杆菌电转化方法的优化

优化了嗜碱芽孢杆菌(Bacillus alcalophilus)TCCC11263的电转化条件,电转化条件包括细胞生长状态、电击场强、电击缓冲液组成、质粒DNA浓度.建立了一种适用于B.alcalophilus TCCC11263的电转化体系.结果为:B alcalophilus TCCC11263培养至OD600为1.0时,电转化效率最高,DNA的转化子数达0.14×103个转化子/μg:用含0.5mol梨醇、0.5mol甘露醇、10%甘油的电击缓冲液洗涤细胞,在场强21kV/cm、电阻200Ω,电容25μF的电击条件下,加入0.7μg质粒DNA,电转化效率达到1.5×103个转化子/μg DNA.     

乳酸乳球菌高效电转化的方法

以质粒pMG36e和pNZ8148为材料,对乳酸乳球ATCC19435的电转化条件进行了优化研究.实验结果表明,乳酸乳球菌的最佳电转化条件为对数生长中后期的细胞,电场强度12 kV/cm,质粒浓度0.1～1 mg/L,复苏时间2 h,此时质粒pMG36e和pNZ8148时供试菌株的电转化效率分别为1.95x105μg-1和2.18x105μg-1,比优化前电转化效率均提高10倍以上.本研究为外源基因电转化乳酸乳球菌提供了可靠的试验参数依据.     

乳杆菌电转化条件的优化

乳杆菌的遗传转化是限制对其遗传操作的关键因素之一.本实验用广宿主质粒pHY300PLK电转化植物乳杆菌(Lactobacillus plantarum)B0080,并优化了转化条件.系统地考察了细胞的生长时期、生长培养基组分、质粒浓度、电击时电场强度、复苏培养基组分等因素对转化效率的影响.选择含5.13%甘氨酸和0.54mol/L蔗糖的MRS培养基制备乳杆菌受体细胞,加入500ng质粒DNA,在电场强度7.41kV/cm电击转化,以含有0.3mol/L蔗糖的MRS培养基复苏培养,获得最佳转化率为3.6×104CFU/μg DNA,比使用初始方法得到的转化率提高了40倍.采用优化后条件,同样实现了pHY300PLK、pBBR1MCS-5对L.amylovoru s B0112和L.acidophilus B0068的高效转化,为进一步对这些菌株进行基因工程改造奠定了有利的方法学基础.     

多黏类芽孢杆菌JSa-9电转化方法的优化

为建立多黏类芽孢杆菌JSa-9菌株的高效电击转化体系,本实验研究菌体培养时间、电场强度、电击缓冲液、质粒用量以及电击后复苏培养时间等因素对JSa-9菌株电转化效率的影响。结果表明：Paenibacillus polymyxaJSa-9培养至OD595 nm为0.3时,电转化效率最高,为0.12×103 CFU/μg DNA;用0.5 mol/L甘露醇、0.5 mol/L山梨醇以及10%甘油的电击缓冲液洗涤细胞,在电场强度17.5 kV/cm、电阻200 Ω,电容25 μF的电击条件下,加入1.0 μg/100 mL质粒DNA,复苏培养时间18 h,电转化效率达到约为0.36×103 CFU/μg DNA;制备P. polymyxa JSa-9原生质体,在电场强度5.0 kV/cm的电击条件下,加入0.8 μg/100 mL质粒DNA,电转化效率较感受态电转提高到约1.0×103 CFU/μg DNA。     

干酪乳杆菌LC2W最优电转化条件的建立

目的建立干酪乳杆菌LC2W电转化的最优条件。方法研究了不同质粒、细胞壁弱化剂浓度、电场强度、复苏时间以及质粒浓度对电转效率的影响,通过单因素轮换法进行了逐一优化。结果经过对乳杆菌感受态制备及电转过程中各因素的考察,得到最佳的参数条件为:添加1%甘氨酸作为细胞壁弱化剂制成感受态细胞,电场强度为10 kV/cm进行高压电击后,复苏3 h,质粒浓度为200 ng/μL时,能够达到最高的电转效率为1.25×106 CFU/μg DNA。结论经优化后,获得了较高的转化水平,可用于指导多种乳杆菌的高效电转,并且为下一步的LC2W菌株改造和基因功能探究奠定了基础。     

嗜热链球菌的电转化条件

为了获得高产活性共轭亚油酸(conjugated linoleic acid,CLA)的嗜热链球菌并开发功能性发酵乳,研究甘氨酸浓度、菌体生物量、电极缓冲液、电压、质粒浓度对电转化效率的影响。结果表明,嗜热链球菌菌体生长与甘氨酸质量浓度呈现负相关,质量浓度为10 g/L时电转化效率最高。电转化效率的影响呈现先上升后下降的趋势;随着菌体生物量、转化电压和质粒浓度的增加,OD600=0. 8、电压1. 8 k V、质粒质量浓度为1. 0 g/L时电转化效率最高;电转缓冲液bufferⅡ(0.5 mol/L蔗糖,1 mmol/L柠檬酸铵; p H 6.0)表现出较好的转化效率。优化嗜热链球菌的电转化条件,提高其转化率,为乳酸菌的高效转化提供一定的理论基础,并为生产具有CLA生理功能的发酵乳提供新的思路。     

干酪乳杆菌LC2W最优电转化条件的建立

目的 建立干酪乳杆菌LC2W电转化的最优条件 。方法 研究了不同质粒、细胞壁弱化剂浓度、电场强度、复苏时间以及质粒浓度对电转效率的影响,通过单因素轮换法进行了逐一优化。结果 经过对乳杆菌感受态制备及电转过程中各因素的考察,得到最佳的参数条件为：添加1 %甘氨酸作为细胞壁弱化剂制成感受态细胞,电场强度为10 kV/cm进行高压电击后,复苏3 h,质粒浓度为200 ng/μL时,能够达到最高的电转效率为1.25×106 CFU/μg DNA。结论 经优化后,获得了较高的转化水平,可用于指导多种乳杆菌的高效电转,并且为下一步的LC2W菌株改造和基因功能探究奠定了基础。     

克雷伯氏肺炎杆菌电击转化条件的优化

针对克雷伯氏肺炎杆菌(Klebsiella pneumoniae)TUAC01的电击转化方法进行了研究。主要考察了培养基EDTA的浓度、细胞的生长状态、感受态细胞的密度、电击电压、质粒浓度等参数对转化效率的影响,建立了一种适合K.pneumoniae TUAC01的电击转化方法。结果:K.pneumoniae TUAC01培养基中添加EDTA至终浓度0.7mmol/L;当细胞生长至OD600为0.7时收集菌体,制作感受态细胞;制作的感受态细胞浓度OD600大于30时,使用2mm的电转杯在2kV的电压下转化,转化效率达到最大值,达到8.58×105cfu/μg DNA。     

构建人Cu,Zn-SOD毕赤酵母转化子及其高表达研究

设计人Cu,Zn-SOD酵母偏爱密码子并化学合成,与pPIC9K连接,构建酵母偏爱密码子的人Cu,Zn-SOD基因真核表达载体,通过电转化和持续加压筛选毕赤酵母GS115高拷贝转化子,获得的重组高表达酵母菌株建立主种子批。经Southern blot鉴定,基因拷贝数提高2～8倍,活性检测显示提高2～4倍。重组菌的目的基因拷贝数与表达产物呈正相关;表达产物为二聚体,其分子质量为40kD左右,低糖基化,均为分泌表达。Western blot法分析,对Cu,Zn-SOD抗体具有特异性反应。转化子在培养16h后进入对数生长期,24h后进入生长稳定期;转化子培养20h左右进行诱导表达最为合适。高拷贝的3株重组菌经50次传代后插入的目的基因保持稳定;Cu,Zn-SOD转化子用正交试验筛选摇瓶的诱导表达条件,经诱导表达,Cu,Zn-SOD表达上清最高活性大于600U/mL。确立最适摇瓶培养条件为pH6.0、30℃、体积分数1.5%甲醇诱导72h测得上清的目的蛋白表达最好,表明构建了高表达菌株。     

Genetic transfer systems for delivery of plasmid deoxyribonucleic acid to Lactobacillus acidophilus ADH: conjugation, electroporation, and transduction

Lactobacillus acidophilus ADH is a bacteriocin-producing human isolate that adheres to human fetal intestinal cells and human ileal cells. We have employed both electroporation and conjugation methodologies to transfer various plasmids to L. acidophilus ADH. Furthermore, we have demonstrated transduction of plasmid DNA within this strain by a temperate bacteriophage (phi adh) harbored by L. acidophilus ADH. Plasmid pGK12 was introduced into strain ADH by electroporation at frequencies as high as 3.3 X 10(5) transformants/micrograms of plasmid DNA. Transconjugants of strain ADH were recovered at frequencies of 10(-2) (pAMB1), 10(-4) (pVA797::Tn917), and 10(-4) (pVA797) per donor cell after filter-mating with Lactococcus lactis ssp. lactis. Plasmid pGK12 was transduced from a phage phi adh lysogen into a recipient strain of L. acidophilus ADH at an average frequency of 3.4 X 10(-8) transductants/pfu. Transformants, transconjugants, or transductants were verified by both phenotype and plasmid profile for acquisition of plasmid DNA. The ability to transfer plasmids and mobilize DNA sequences by electroporation, conjugation, and transduction will augment our efforts to define and characterize the activities of L. acidophilus in the intestinal tract.     

植物乳杆菌G63电转化方法的优化

为获得植物乳杆菌G63高效的电转化方法,本研究从细胞生长状态、细胞弱化剂质量浓度、洗涤液、质粒添加量和电击参数等方面对菌株G63的电转化效率进行优化。结果表明：取菌株G63对数生长中期的细胞制备感受态,以1 g/100 mL甘氨酸作为细胞弱化剂,分别用1 mmol/L MgCl2和30 g/100 mL聚乙二醇1000洗涤细胞,并用30 g/100 mL聚乙二醇1000作为电击液,加入20 μg穿梭质粒,在1.5 kV和400 Ω条件下进行电击,可以获得最高的电转化效率,转化效率达到1.18×103 CFU/μg DNA,满足后续遗传学实验要求。     

Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus

Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4⁺ of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early‐log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4⁺ probe. The plasmid containing ade6⁺ suppressed the auxotrophic growth of the ade6‐domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus. RS1 has been submitted to the DDBJ/EMBL/GenBank database (Accession No. AB547343). Copyright © 2010 John Wiley & Sons, Ltd.     

Convenient transformation of anamorphic basidiomycetous yeasts belonging to genus pseudozyma induced by electroporation

A convenient procedure for carrying out transformation by electroporation was optimized for the genus Pseudozyma. Successful transformation was achieved using a plasmid, pUXV1, that confers resistance to hygromycin B; the maximum transformation efficiency was 48 transformants/mug of plasmid DNA. Transformants of Pseudozyma antarctica T-34 expressing a green fluorescent protein were obtained by the procedure.     

解淀粉芽孢杆菌psd基因过表达对胞苷发酵的影响

以解淀粉芽孢杆菌BG-09(Bacillus amyloliquefaciens BG-09)为出发菌株,分析代谢途径中与细胞膜渗透性有关的基因psd,研究过表达基因psd对胞苷发酵的影响。以解淀粉芽孢杆菌BG-09基因组为模板克隆psd,构建了含psd的重组质粒pHT43-psd,通过化学转化法转入菌株大肠杆菌DH5α,验证成功后将其转入B.amyloliquefaciens BG-09,获得重组菌株B.amyloliquefaciens BG-09-psd。将B.amyloliquefaciens BG-09-psd菌株通过摇瓶发酵,研究过表达基因psd对菌体的生长、胞苷和尿苷产量积累的影响。结果显示,重组菌株B.amyloliquefaciens BG-09-psd发酵液中胞苷浓度为1.199 g/L,与对照菌株B.amyloliquefaciens BG-09相比,提高了15.51%,尿苷浓度为0.552 g/L,增加了6.56%,表明过表达基因psd可促进胞苷的积累。     

Efficient electropulse transformation of intact Candida maltosa cells by different homologous vector plasmids.

Conditions for efficient and quick transformation by electroporation were developed in Candida maltosa. To investigate the efficiency of transformation with integrative as well as with autonomously replicating plasmids, a series of vectors was constructed for homologous transformation of this species. Transformants were obtained with different plasmids as covalently closed circular molecules and as linearized DNA. The influence of recipient strain and plasmid type as well as of cell number and parameters of the supplied electrical pulse on the transformation efficiency have been investigated. A maximum of 7000 transformants per 100 ng of plasmid DNA was reached. The efficiency of transformation was compared with that of the LiCl method.     

Transformation of Candida albicans by electroporation

In contrast to a variety of other yeasts, Candida albicans has proved difficult to transform with high efficiency. Lithium acetate transformation is fast and simple but provides a very low efficiency of DNA transfer (50-100 transformants/microg DNA), while spheroplast transformation, although more efficient ( approximately 300 transformants/microg integrative DNA and 10(3)-10(4) transformants/microg replicative DNA), is complicated and time-consuming. In this study we applied various yeast transformation techniques to C. albicans and selected an electroporation procedure for further optimization. Transformation efficiencies of up to 300 transformants/microg were obtained for an integrative plasmid and up to 4500 transformants/microg for a CARS-carrying plasmid. This reasonably high transformation efficiency, combined with the ease and speed of electroporation in comparison to alternative techniques, make it the preferred method for transformation of C. albicans.