Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis

Tran, Dinh T.; Phan, Trang Thi Phuong; Huynh, Thanh Kieu; Dang, Ngan Thi Kim; Huynh, Phuong Truc; Nguyen, Tri Khoa; Truong, Tuom Thi Tinh; Tran, Thuoc Linh; Schumann, Wolfgang; Nguyen, Hoang

doi:10.1186/s12934-017-0747-0

Cited by 32 publications

(23 citation statements)

References 19 publications

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“…We first created the three basic inducer-free integrative expression vectors, pHT2171, pHT2184 and pHT2188, and then, the bgaB gene was introduced into these vectors to obtain pHT2172, pHT2185 and pHT2189. pHT2171 was constructed by inserting the cassette containing the lacO3 operator and the P grac 01 promoter (amplified from pHT2134 as the template, a derivative of pHT2071 [ 19 ] carrying a new MCS, with ON2195/ON2194) into the pHT1305 empty vector which contains sequences to allow integration at the lacA locus on the B. subtilis genome without any promoter. pHT2184 was constructed by insertion of the cassette harboring lacO3 and the P grac 100 promoter into the pHT1326 backbone.…”

Section: Methodsmentioning

confidence: 99%

“…Samples were prepared for activity measurements and for SDS-PAGE analysis. BgaB activity was measured as described in [ 19 ].…”

Section: Methodsmentioning

confidence: 99%

“…The unsolved problem in the generation of useful vectors allowing robust B. subtilis expression systems is their leaky expression in E. coli. Most of the expression vectors for B. subtilis are shuttle-vectors because the cloning steps must be carried out in E. coli [ 3 ], which can sometimes result in unexpectedly high protein expression levels [ 15 , 16 ], and, in the worst case, kill the E. coli cells. Previous publications did not mention whether strong promoters for B. subtilis also cause high background expression in E. coli.…”

Section: Introductionmentioning

confidence: 99%

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Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis

Tran

Phan

Doan

et al. 2020

Biotechnology Reports

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Highlights The new inducer-free integrative expression vectors could repress the reporter gene expression in the E. coli cloning strain, thereby facilitating the cloning step. The expression vectors carrying IPTG-inducible P grac promoters allow the production of the recombinant protein at high levels in B. subtilis in the absence of the inducer. The single-copy expression levels of integrative constructs, P grac 01- bgaB , P grac 100- bgaB , P grac 212- bgaB could reach to % and 8%, 20.9 % and 42 % of total cellular proteins after 12 h incubation, respectively. The double integration of P grac 212- bgaB into both amyE and lacA loci resulted in BgaB expression up to 53.4 %.

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Section: Methodsmentioning

confidence: 99%

“…Samples were prepared for activity measurements and for SDS-PAGE analysis. BgaB activity was measured as described in [ 19 ].…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis

Tran

Phan

Doan

et al. 2020

Biotechnology Reports

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“…B. subtilis 1012 strains carrying the recombinant plasmids pHT2002 and pHT1512 (negative) was cultured in an LB medium containing the appropriate antibiotic. We followed the protocol as described elsewhere 10 . Briefly, the bacterial cells were grown to the midlogarithmic growth phase and then induced with 0.1 mM, 1 mM IPTG.…”

Section: Expression Of the Reporter β -Galactosidasementioning

confidence: 99%

Construction of expression plasmid for Bacillus subtilis using Pspac promoter and BgaB as a reporter

et al. 2019

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Introduction:In basic research, it is essential to use an inducible promoter which can be controlled to express a small amount of protein for studying their roles in the cell. Pspac, a well-known weak promoter for Bacillus subtilis, uses isopropyl β -D-1-thiogalactopyranoside (IPTG) as an inducer. However, plasmids carrying this promoter such as pHCMC05 still have a disadvantage which harbors a repetitive DNA fragment of about 200 bp, resulting in structural instability in Escherichia coli, causing difficulty during cloning. Methods: In this study, we constructed a plasmid that does not carry the repetitive sequences and investigated plasmid structural stability in E. coli, then measured the β -galactosidase reporter gene (bgaB) expression in B. subtilis. Results: The constructed plasmid pHT2002 was stable over 56 generations while pHCMC05-bgaB was structurally instable and ultimately lost after 42 generations. BgaB activities and Western-blot indicated that BgaB-coding gene under control of IPTG-inducible promoter Pspac could be expressed at low levels. Conclusion: The study demonstrated that the new expression plasmid without the repeated sequences retained its structural stability in E. coli facilitating the cloning step. The expression plasmid with Pspac promoter for B. subtilis could be used to express a modest amount of the heterologous protein in the presence of IPTG.

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“…Expression of proteins still requires tight control, relevant for toxic proteins, which is disadvantageous in induction-free plasmids [ 23 ].…”

Section: Introductionmentioning

confidence: 99%

Replacing Standard Reporters from Molecular Cloning Plasmids with Chromoproteins for Positive Clone Selection

et al. 2018

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Cloning and expression plasmids are the workhorses of modern molecular biology. Despite the pathway paved by synthetic biology, laboratories around the globe still relay on standard cloning techniques using plasmids with reporter proteins for positive clone selection, such as β-galactosidase alpha peptide complementation for blue/white screening or ccdB, which encodes for a toxic DNA gyrase. These reporters, when interrupted, serve as a positive clone detection system. In the present report, we show that molecular cloning plasmids bearing the coding sequence for a 25.4 kDa protein, AmilCP, encoded by a 685 bp gene, that is well expressed in Escherichia coli, render blue-purple colonies. Using this reporter protein, we developed and tested a cloning system based on the constitutive expression of the non-toxic AmilCP protein, that once interrupted, the loss of purple color serves to facilitate positive clone selection. The main advantage of this system is that is less expensive than other systems since media do not contain chromogenic markers such as X-gal, which is both expensive and cumbersome to prepare and use, or inductors such as IPTG. We also designed an inducible expression plasmid suitable for recombinant protein expression that also contains AmilCP cloning selection marker, a feature not commonly found in protein expression plasmids. The use of chromogenic reporters opens an important avenue for its application in other organisms besides E. coli for clone selection or even for mutant selection.

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Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis

Cited by 32 publications

References 19 publications

Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis

Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis

Construction of expression plasmid for Bacillus subtilis using Pspac promoter and BgaB as a reporter

Replacing Standard Reporters from Molecular Cloning Plasmids with Chromoproteins for Positive Clone Selection

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