Construction of pSETsac B plasmid and evaluation of kdp E gene knockout efficiency with and without markers in Aeromonas dhakensis

LING Bo; CHEN Liqing; WANG Yu; YANG Nuo; FAN Lixia; GUO Guiying; LI Xuesong; CENG Jifeng; LI Qian; ZHENG Jiping

doi:10.15886/j.cnki.rdswxb.20230056

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Volume 15 Issue 4

Jul. 2024

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Article Navigation > Journal of Tropical Biology > 2024 > 15(4): 452-459

LING Bo, CHEN Liqing, WANG Yu, YANG Nuo, FAN Lixia, GUO Guiying, LI Xuesong, CENG Jifeng, LI Qian, ZHENG Jiping. Construction of pSETsac B plasmid and evaluation of kdp E gene knockout efficiency with and without markers in Aeromonas dhakensis[J]. Journal of Tropical Biology, 2024, 15(4): 452-459. doi: 10.15886/j.cnki.rdswxb.20230056

Citation:

LING Bo, CHEN Liqing, WANG Yu, YANG Nuo, FAN Lixia, GUO Guiying, LI Xuesong, CENG Jifeng, LI Qian, ZHENG Jiping. Construction of pSETsac B plasmid and evaluation of kdp E gene knockout efficiency with and without markers in Aeromonas dhakensis[J]. Journal of Tropical Biology, 2024, 15(4): 452-459. doi: 10.15886/j.cnki.rdswxb.20230056

Construction of pSETsac B plasmid and evaluation of kdp E gene knockout efficiency with and without markers in Aeromonas dhakensis

doi: 10.15886/j.cnki.rdswxb.20230056

1. School of Animal Science and Technology, Hainan University, Haikou, Hainan 570228;
2. Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106;
3. School of Life Sciences, Hainan University, Haikou, Hainan 570228;
4. School of Science, Hainan University, Haikou, Hainan 570228;
5. Network and Technology Center, Hainan University, Haikou, Hainan 570228, China

Received Date: 2023-04-26
Rev Recd Date: 2023-09-20
Publish Date: 2024-07-25

Abstract

In order to construct a novel gene-knockout plasmid, a novel plasmid pSETsac B was constructed based on the plasmids pSET4s and pK18mobsac B by using PCR and cloning techniques. Then pK18mobsac B was used as control, the knockout efficiency of kdp E gene in Aeromonas dhakensis was evaluated via the newly constructed plasmid with or without tet^r marker. The results showed that there was no difference in the knockout efficiency between the two plasmids with tet^r marker, but the efficiency of scarless deletion by pSETsac B was significantly higher than that of the control plasmid pK18mobsac B. p SETsac B is a thermosensitive shuttle plasmid, and is hence expected to play a role in the genetic mechanism of Gram-positive and Gram-negative bacteria. This study also provides important mutant material for investigating the kdp E gene of A. dhakensis.
- pSETsac B,
- kdp E gene,
- gene knockout

References

[1]	KITAGAWA H, OHGE H, YU L, et al. Aeromonas dhakensis is not a rare cause of Aeromonas bacteremia in Hiroshima, Japan[J]. Journal of Infection and Chemotherapy:Official Journal of the Japan Society of Chemotherapy,2020, 26(2):316-320.
[2]	CHEN P L, WU C J, CHEN C S, et al. A comparative study of clinical Aeromonas dhakensis and Aeromonas hydrophila isolates in southern Taiwan:A. dhakensis is more predominant and virulent[J]. Clinical Microbiology and Infection, 2014, 20(7):O428-O434.
[3]	HEERMANN R, JUNG K. The complexity of the ‘simple’two-component system KdpD/KdpE in Escherichia coli[J].FEMS Microbiology Letters, 2010, 304(2):97-106.
[4]	FREEMAN Z N, DORUS S, WATERFIELD N R. The Kdp D/kdp E two-component system:integrating K+homeostasis and virulence[J]. PLoS Pathogens, 2013, 9(3):e1003201.
[5]	丁海峰,李小申,黄慧,等.基因敲除技术研究进展[J].黑龙江畜牧兽医, 2018(5):50-54.
[6]	WANG C, SUN H. Progress in gene knockout mice[J].Sheng Wu Gong Cheng Xue Bao, 2019, 35(5):784-794.
[7]	LIU Z, LIU P, LIU S, et al. Small protein B upregulates sensor kinase bvg S expression in Aeromonas veronii[J].Frontiers in Microbiology, 2015, 16:6:579.
[8]	SONG T, TOMA C, NAKASONE N, et al. Aerolysin is activated by metalloprotease in Aeromonas veronii biovar sobria[J]. Journal of Medical Microbiology, 2004, 53(6):477-482.
[9]	SILVER A C, RABINOWITZ N M, KUF FER S, et al.Identification of Aeromonas veronii genes required for colonization of the medicinal leech, Hirudo verbana[J]. Journal of Bacteriology, 2007, 189(19):6763-6772.
[10]	WEI Z, FU Q, CHEN Y, et al. The capsule of Streptococcus equi ssp. zooepidemicus is a target for attenuation in vaccine development[J]. Vaccine, 2012, 30(31):4670-4675.
[11]	QUÉNÉE L, LAMOTTE D, POLACK B. Combined sacBbased negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in Pseudomonas aeruginosa[J]. BioTechniques, 2005, 38(1):63-67.
[12]	AMBROSIS N, FERNÁNDEZ J, SISTI F. Counterselection method for markerless allelic exchange in Bordetella bronchiseptica based on sac B gene from Bacillus subtilis[J]. Current Protocols in Microbiology, 2020, 59(1):e125.
[13]	TAKAMATSU D, OSAKI M, SEKIZAKI T. Thermosensitive suicide vectors for gene replacement in Streptococcus suis[J]. Plasmid, 2001, 46(2):140-148.
[14]	常雅洁,丁雪燕,王思权,等.常见细菌基因敲除技术的研究进展[J].中国预防兽医学报, 2021, 43(10):1118-1123.
[15]	李颖平,张映,邵国青.一种新的基因敲除术-RNAi[J].生物技术通报, 2006(3):42-45.
[16]	李今煜,陈健铭,彭振坤.几种常用的基因敲除技术[J].武夷科学, 2007, 23(0):187-190.
[17]	MILLER J C, HOLMES M C, WANG J, et al. An improved zinc-finger nuclease architecture for highly specific genome editing[J]. Nature Biotechnology, 2007,25(7):778-785.
[18]	GAJ T, GERSBACH C A, BARBAS III C F. ZFN,TALEN, and CRISPR/Cas-based methods for genome engineering[J]. Trends in Biotechnology, 2013, 31(7):397-405.
[19]	JOUNG J, KONERMANN S, GOOTENBERG J S, et al.Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening[J]. Nature Protocols, 2017, 12(4):828-863.
[20]	DALVIE N C, LORGEREE T, BIEDERMANN A M, et al. Simplified gene knockout by CRISPR-Cas9-induced homologous recombination[J]. ACS Synthetic Biology,2022, 11(1):497-501.
[21]	ALI M K, LI X, TANG Q, et al. Regulation of inducible potassium transporter KdpFABC by the KdpD/KdpE two-component system in Mycobacterium smegmatis[J].Frontiers in Microbiology, 2017, 8:570.
[22]	余华,熊浚智,何晓梅,等.采用Red重组系统敲除铜绿假单胞菌弹性蛋白酶基因[J].中国人兽共患病学报, 2013, 29(2):129-132.
[23]	LIU R, ZHANG P, SU Y, et al. A novel suicide shuttle plasmid for Streptococcus suis serotype 2 and Streptococcus equi ssp. zooepidemicus gene mutation[J]. Scientific Reports, 2016, 6:27133.
[24]	吴菁,刘秀敏,张维,等.枯草芽孢杆菌sac B基因的功能验证及应用[J].核农学报, 2008, 22(5):590-594.
[25]	SUN X, YANG D, WANG Y, et al. Development of a markerless gene deletion system for Streptococcus zooepidemicus:functional characterization of hyaluronan synthase gene[J]. Applied Microbiology and Biotechnology,2013, 97(19):8629-8636.
[26]	HOOVEN T A, BONAKDAR M, CHAMBY A B, et al. A counterselectable sucrose sensitivity marker permits efficient and flexible mutagenesis in Streptococcus agalactiae[J]. Applied and Environmental Microbiology, 2019,85(7):e03009-e03018.
[27]	SCHÄFER A, TAUCH A, JÄGER W, et al. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum[J]. Gene, 1994, 145(1):69-73.

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通讯作者: 陈斌, bchen63@163.com

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沈阳化工大学材料科学与工程学院沈阳 110142

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Construction of pSETsac B plasmid and evaluation of kdp E gene knockout efficiency with and without markers in Aeromonas dhakensis

1. School of Animal Science and Technology, Hainan University, Haikou, Hainan 570228;

2. Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106;

3. School of Life Sciences, Hainan University, Haikou, Hainan 570228;

4. School of Science, Hainan University, Haikou, Hainan 570228;

5. Network and Technology Center, Hainan University, Haikou, Hainan 570228, China

Received Date: 2023-04-26

Rev Recd Date: 2023-09-20

Publish Date: 2024-07-25

Key words:

pSETsac B /
kdp E gene /
gene knockout

Abstract: In order to construct a novel gene-knockout plasmid, a novel plasmid pSETsac B was constructed based on the plasmids pSET4s and pK18mobsac B by using PCR and cloning techniques. Then pK18mobsac B was used as control, the knockout efficiency of kdp E gene in Aeromonas dhakensis was evaluated via the newly constructed plasmid with or without tet^r marker. The results showed that there was no difference in the knockout efficiency between the two plasmids with tet^r marker, but the efficiency of scarless deletion by pSETsac B was significantly higher than that of the control plasmid pK18mobsac B. p SETsac B is a thermosensitive shuttle plasmid, and is hence expected to play a role in the genetic mechanism of Gram-positive and Gram-negative bacteria. This study also provides important mutant material for investigating the kdp E gene of A. dhakensis.

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Reference (27)

[1]	KITAGAWA H, OHGE H, YU L, et al. Aeromonas dhakensis is not a rare cause of Aeromonas bacteremia in Hiroshima, Japan[J]. Journal of Infection and Chemotherapy:Official Journal of the Japan Society of Chemotherapy,2020, 26(2):316-320.
[2]	CHEN P L, WU C J, CHEN C S, et al. A comparative study of clinical Aeromonas dhakensis and Aeromonas hydrophila isolates in southern Taiwan:A. dhakensis is more predominant and virulent[J]. Clinical Microbiology and Infection, 2014, 20(7):O428-O434.
[3]	HEERMANN R, JUNG K. The complexity of the ‘simple’two-component system KdpD/KdpE in Escherichia coli[J].FEMS Microbiology Letters, 2010, 304(2):97-106.
[4]	FREEMAN Z N, DORUS S, WATERFIELD N R. The Kdp D/kdp E two-component system:integrating K+homeostasis and virulence[J]. PLoS Pathogens, 2013, 9(3):e1003201.
[5]	丁海峰,李小申,黄慧,等.基因敲除技术研究进展[J].黑龙江畜牧兽医, 2018(5):50-54.
[6]	WANG C, SUN H. Progress in gene knockout mice[J].Sheng Wu Gong Cheng Xue Bao, 2019, 35(5):784-794.
[7]	LIU Z, LIU P, LIU S, et al. Small protein B upregulates sensor kinase bvg S expression in Aeromonas veronii[J].Frontiers in Microbiology, 2015, 16:6:579.
[8]	SONG T, TOMA C, NAKASONE N, et al. Aerolysin is activated by metalloprotease in Aeromonas veronii biovar sobria[J]. Journal of Medical Microbiology, 2004, 53(6):477-482.
[9]	SILVER A C, RABINOWITZ N M, KUF FER S, et al.Identification of Aeromonas veronii genes required for colonization of the medicinal leech, Hirudo verbana[J]. Journal of Bacteriology, 2007, 189(19):6763-6772.
[10]	WEI Z, FU Q, CHEN Y, et al. The capsule of Streptococcus equi ssp. zooepidemicus is a target for attenuation in vaccine development[J]. Vaccine, 2012, 30(31):4670-4675.
[11]	QUÉNÉE L, LAMOTTE D, POLACK B. Combined sacBbased negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in Pseudomonas aeruginosa[J]. BioTechniques, 2005, 38(1):63-67.
[12]	AMBROSIS N, FERNÁNDEZ J, SISTI F. Counterselection method for markerless allelic exchange in Bordetella bronchiseptica based on sac B gene from Bacillus subtilis[J]. Current Protocols in Microbiology, 2020, 59(1):e125.
[13]	TAKAMATSU D, OSAKI M, SEKIZAKI T. Thermosensitive suicide vectors for gene replacement in Streptococcus suis[J]. Plasmid, 2001, 46(2):140-148.
[14]	常雅洁,丁雪燕,王思权,等.常见细菌基因敲除技术的研究进展[J].中国预防兽医学报, 2021, 43(10):1118-1123.
[15]	李颖平,张映,邵国青.一种新的基因敲除术-RNAi[J].生物技术通报, 2006(3):42-45.
[16]	李今煜,陈健铭,彭振坤.几种常用的基因敲除技术[J].武夷科学, 2007, 23(0):187-190.
[17]	MILLER J C, HOLMES M C, WANG J, et al. An improved zinc-finger nuclease architecture for highly specific genome editing[J]. Nature Biotechnology, 2007,25(7):778-785.
[18]	GAJ T, GERSBACH C A, BARBAS III C F. ZFN,TALEN, and CRISPR/Cas-based methods for genome engineering[J]. Trends in Biotechnology, 2013, 31(7):397-405.
[19]	JOUNG J, KONERMANN S, GOOTENBERG J S, et al.Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening[J]. Nature Protocols, 2017, 12(4):828-863.
[20]	DALVIE N C, LORGEREE T, BIEDERMANN A M, et al. Simplified gene knockout by CRISPR-Cas9-induced homologous recombination[J]. ACS Synthetic Biology,2022, 11(1):497-501.
[21]	ALI M K, LI X, TANG Q, et al. Regulation of inducible potassium transporter KdpFABC by the KdpD/KdpE two-component system in Mycobacterium smegmatis[J].Frontiers in Microbiology, 2017, 8:570.
[22]	余华,熊浚智,何晓梅,等.采用Red重组系统敲除铜绿假单胞菌弹性蛋白酶基因[J].中国人兽共患病学报, 2013, 29(2):129-132.
[23]	LIU R, ZHANG P, SU Y, et al. A novel suicide shuttle plasmid for Streptococcus suis serotype 2 and Streptococcus equi ssp. zooepidemicus gene mutation[J]. Scientific Reports, 2016, 6:27133.
[24]	吴菁,刘秀敏,张维,等.枯草芽孢杆菌sac B基因的功能验证及应用[J].核农学报, 2008, 22(5):590-594.
[25]	SUN X, YANG D, WANG Y, et al. Development of a markerless gene deletion system for Streptococcus zooepidemicus:functional characterization of hyaluronan synthase gene[J]. Applied Microbiology and Biotechnology,2013, 97(19):8629-8636.
[26]	HOOVEN T A, BONAKDAR M, CHAMBY A B, et al. A counterselectable sucrose sensitivity marker permits efficient and flexible mutagenesis in Streptococcus agalactiae[J]. Applied and Environmental Microbiology, 2019,85(7):e03009-e03018.
[27]	SCHÄFER A, TAUCH A, JÄGER W, et al. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19:selection of defined deletions in the chromosome of Corynebacterium glutamicum[J]. Gene, 1994, 145(1):69-73.

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Construction of pSETsac B plasmid and evaluation of kdp E gene knockout efficiency with and without markers in Aeromonas dhakensis

doi: 10.15886/j.cnki.rdswxb.20230056

Abstract

References

Proportional views

通讯作者: 陈斌, bchen63@163.com

Proportional views

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