| 主办单位: |
| 中国科学院遗传与发育生物学研究所 |
| 中国遗传学会 |
| 编辑单位: |
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《遗传学报》 编辑部
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| 主 编: 薛勇彪 |
| 主 任: 张颖 |
编委阵容 |
| 审稿专家 |
| 国内刊号： |
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CN 11-5450/R
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| 国际刊号： |
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ISSN 1673-8527
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| 邮发代号： 2-819
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遗传学报 2004.3 Vol 31, No.3 311-316
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| 铜绿假单胞菌最佳电转化条件的研究 |
| The Study of Optimal Conditions of Electroporation in Pseudomonas aeruginosa |
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| 单志英，徐海津，施兴启，聂 舟，遇 言，张秀明，白艳玲，乔明强①，高才昌 |
| 南开大学分子生物学研究所，天津 300071 |
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| Key Words： 铜绿假单胞菌；电转化；Mu转座复合物 |
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Abstract
以临床分离的一株铜绿假单胞菌(Pseudomonas aeruginos a)PA68作受体菌，将 具有卡那霉素抗性标记的质粒pSMC28通过电转化导入到受体菌中，研究细胞生长状态、电击 电压、细胞浓度、感受态细胞的贮备方式对转化效率的影响。结果表明，在细胞生长至OD 540 =0.7～0.8时收集菌体，在低温(2℃)条件下，制备浓度为10 11 个细胞/ mL的感受态细胞，在较高的电压(2.6 kV)电击下，能获得较高的转化效率。最高可达1.68 ×108个转化子/μg DNA（CFU/μg DNA）。用此优化的转化条件，在国际上首次成功地将 Mu转座复合物导入到P.aeruginosa中，并获得2.4×104 CFU/μg DNA的高转化效率 。由于Mu转座重组技术具有随机单点插入的优点，克服了传统转座子能在染色体上迁移的缺 点，保证了表型的改变与转座子插入位点所在的基因突变的一一对应关系，为进一步研究 P.aeruginosa的基因组功能奠定基础。 |
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Abstract
A P.aeruginosa strain PA68 isolated from the sp utum of a patient suffering from bronchiectasis was used as the recipient strain .Optimum conditions including growth stage of the strain,electroshock voltage,co ncentration and preservation of competent cell were defined for the electroporat ion of PA68 with plasmid pSMC28.It was showed that the highest transformation ef ficiency was up to 1.68×108 CFU/μg DNA under the optimium conditions in whi ch the competent cells were collected at logarithmic growth phase (OD 540 =0.7～0.8) and concentrated to about 10 11 cells/ml,the mixture of the competent cells and plasmid pSMC28 was eletroporated at 2.6 kV.With this optim al condition,Mu transponson complexes have been successfully transformed into P.aeruginosa strain PA68 and the obtained efficiency was 2.47×104 CFU/μg DNA.This is the first time to electroporate Mu trsnsposon complexes into Pseudomon as spp.The artificial Mu transposons could integrate into bacterial geno mes at a single site randomly.T hen the phenotype change was the result of the gene inactivation caused by Mu tr ansposon insertion.That will be very helpful for the study of genomic function of Pseudomonas spp. |
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中国科学院遗传与发育生物学研究所
