A new method for multiple gene inactivations in Bacillus subtilis 168, producing a strain free of selectable markers

2011 ◽  
Vol 57 (5) ◽  
pp. 427-436 ◽  
Author(s):  
Chong Zhang ◽  
Xiaohui Zhang ◽  
Zhengying Yao ◽  
Yaping Lu ◽  
Fengxia Lu ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Resistance Gene ◽  
Target Genes ◽  
Genome Structure ◽  
Subtilis Strain ◽  
Gene Target ◽  
Selectable Markers ◽  
Vector Integration ◽  
Bacillus Subtilis 168 ◽  
Counter Selection

This study describes a novel method for repeated gene inactivation in Bacillus subtilis 168. A B. subtilis strain (BS-PS) that is conditionally auxotrophic for lysine was obtained by replacing the PlysA promoter with the Pspac promoter. The homologous recombination integration vector PLC-T was constructed to contain lacI, which encodes a Pspac promoter repressor, and the chloromycetin resistance gene. Target genes were manipulated by generating an insertion sequence with two homologous arms and the target gene in PLC-T to create a specific integrating vector. Integration into the BS-PS chromosome occurred by a single crossover at either of the two homologous arms. The resulting transitional strain (BS-PS-PI) was chloromycetin resistant and lysine auxotrophic and had an unstable genome structure because of the duplication. Excision of lacI and chloromycetin resistance gene was achieved by a second single crossover at the duplication. Recovery of a lysine prototroph functioned as counter-selection and was identified by PCR. In this work, we inactivated nprE and aprE, two protease genes secreted by B. subtilis 168 free of selectable markers.

scholarly journals Improving the Production of Salt-Tolerant Glutaminase by Integrating Multiple Copies of Mglu into the Protease and 16S rDNA Genes of Bacillus subtilis 168

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 592 ◽  
Author(s):  
Xian Zhang ◽  
Zhaoyang Xu ◽  
Song Liu ◽  
Kai Qian ◽  
Meijuan Xu ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
16S Rdna ◽  
Micrococcus Luteus ◽  
Rdna Locus ◽  
Subtilis Strain ◽  
Enzyme Activity Assay ◽  
Bacillus Subtilis 168 ◽  
Fed Batch Fermentation ◽  
Multiple Copies

In this study, the Micrococcus luteus K-3 glutaminase was successfully over-expressed in the GRAS (Generally Recognized as Safe) Bacillus subtilis strain 168 by integration of the Mglu gene in the 16S rDNA locus. This was done in order to screen a strain producing high levels of recombinant glutaminase from the selected candidates. The transcription of the glutaminase genes in the B. subtilis 168 chromosome and the expression of glutaminase protein was further assessed by qPCR, SDS-PAGE analysis and an enzyme activity assay. To further increase the production of glutaminase, the nprB and nprE genes, which encode specific proteases, were disrupted by integration of the Mglu gene. After continuous cell culturing without the addition of antibiotics, the integrated recombinant strains showed excellent genetic stability, demonstrating favorable industrialization potential. After the fermentation temperature was optimized, a 5-L bioreactor was used for fed-batch fermentation of the recombinant glutaminase producing strain at 24 °C, and the highest enzyme activity achieved was approximately 357.6 U/mL.

scholarly journals Pencarian Alel untuk Identifikasi Gen Ketahanan Penyakit Hawar Daun Bakteri, Xa7 pada Plasma Nutfah Padi Lokal Indonesia

2016 ◽  
Vol 6 (1) ◽  
pp. 1 ◽  
Author(s):  
Dwinita W Utami ◽  
Endang M Septiningsih ◽  
Triny S Kadir ◽  
Fatimah W Fatimah ◽  
Siti W Yuriyah
Keyword(s):  
Genetic Diversity ◽  
Resistance Gene ◽  
Target Genes ◽  
Rice Genome ◽  
Superior Performance ◽  
Allele Mining ◽  
Gene Target ◽  
Rice Germplasm ◽  
Germplasm Collections ◽  
Plant Breeding Program

<p>Allele Mining of Bacterial Blight Resistance Gene, Xa7 on<br />Indonesian Local Rice Germplasm. Dwinita W. Utami,<br />Endang M. Septiningsih, Trini S. Kadir, Fatimah, and Siti<br />Yuriyah. The abundance of novel genetic variation existing<br />in germplasm collections is the foundation for variety<br />improvement in plant breeding program. Nevertheless,<br />studies on Indonesian genetic diversity rice germplasm<br />using molecular markers are still poorly. Recent advances in<br />utilizing of simple sequence repeat (SSR) in QTL mapping<br />and whole rice genome sequences were positive support on<br />genetic diversity of rice germplasm research. Based on these<br />advance technology, we developed the research to discover<br />new alleles at important gene loci that can be used for rice<br />improvement. This approach is recognized as allele mining<br />technology. On this study the target genes for allele mining<br />research is the resistance gene for bacterial leaf blight<br />pathogen, Xa7. This point was introduced by identify the<br />genetic diversity of 96 accessions Indonesian local rice<br />germplasm. The Xa7 allele mining was done by SNP (single<br />nucleotide polymorphism) designing primers based on DNA<br />sequence around the gene target. The significant LD map<br />was detected by association mapping between phenotype<br />and SNP genotyping data of the selected germplasm which<br />having superior performance on BLB resistance and<br />representing on genetic diversity clustering. The results<br />shown that Xa7 allele variation were found in Parekaligolara<br />(Indica, 15141), and Gajah Mada (Indica, 5856), which<br />resistant to BLB races IV and VIII on generative stage and<br />field condition. The significant Xa7-SNP8 and Xa7-SNP11<br />markers were associating with the LD map position of Xa7<br />gene on 28, 05-28,1Mb of chromosome 6 in rice genome.</p>

Crystal Structure of Urate Oxidase from Bacillus Subtilis 168

2019 ◽  
Vol 64 (7) ◽  
pp. 1126-1133 ◽  
Author(s):  
A. Nayab ◽  
S. A. Moududee ◽  
Y. Shi ◽  
Y. Jiang ◽  
Q. Gong
Keyword(s):  
Crystal Structure ◽  
Bacillus Subtilis ◽  
Urate Oxidase ◽  
Bacillus Subtilis 168
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