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Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations.

Wegerer A, Sun T, Altenbuchner J - BMC Biotechnol. (2008)

Bottom Line: The introduction of a stem loop into the translation initiation region of the rhaPBAD promoter resulted in the most significant improvement of eGFP expression.The plasmid pWA21, containing the stem loop, one cer site and rop, attained high expression levels accompanied by a good stability, and on that score seems to be a well-balanced choice.The genetic modules showed a complex interplay, therefore two positive effects combined sometimes resulted in a disadvantage.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. a_wegerer@yahoo.de

ABSTRACT

Background: A capable expression vector is mainly characterized by its production efficiency, stability and induction response. These features can be influenced by a variation of modifications and versatile genetic modules.

Results: We examined miscellaneous variations of a rhaPBAD expression vector. The introduction of a stem loop into the translation initiation region of the rhaPBAD promoter resulted in the most significant improvement of eGFP expression. Starting from this plasmid, we constructed a set of expression vectors bearing different genetic modules like rop, ccdAB, cer and combinations thereof, and tested the efficiency of expression and plasmid stability. The plasmid pWA21, containing the stem loop, one cer site and rop, attained high expression levels accompanied by a good stability, and on that score seems to be a well-balanced choice.

Conclusion: We report the generation of variations of the rhaPBAD expression vector and characterization hereof. The genetic modules showed a complex interplay, therefore two positive effects combined sometimes resulted in a disadvantage.

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Related in: MedlinePlus

Plasmid stability after 48 h in liquid culture without antibiotic selection. Cells were grown at 37°C in LB supplemented with 0.2% (w/v) L-rhamnose for 24 h, starting from this culture fresh medium was inoculated, and again incubated for 24 h under the same conditions, which roughly matches 50 generations. The percentage of cells without plasmid or loss of fluorescence are shown, the values are the averages of three independent experiments.
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Figure 4: Plasmid stability after 48 h in liquid culture without antibiotic selection. Cells were grown at 37°C in LB supplemented with 0.2% (w/v) L-rhamnose for 24 h, starting from this culture fresh medium was inoculated, and again incubated for 24 h under the same conditions, which roughly matches 50 generations. The percentage of cells without plasmid or loss of fluorescence are shown, the values are the averages of three independent experiments.

Mentions: The usability of a plasmid is not only determined by the amount of protein produced in a specific combination of strain, gene and plasmid, but also by the stability of the expression vector. Especially if the induction is carried out on a large scale and during a longer period of process, plasmid loss can have a tremendous effect on total yield. In order to test the influence of the variations of the genetic modules, we determined how many cells lost their plasmids during a prolonged induction. If the cultures were induced at 30°C for 48 or 96 hours, which is roughly equivalent to 50 generations or 100 generations, respectively, no plasmid loss could be observed in none of the cultures (data not shown). But if the induction was carried out at 37°C for 48 hours, major differences between the plasmids could be detected. The originating pJOE4056.2 and its two direct derivatives pJOE5058.1 and pWA19 were perfectly stable (Fig. 4). These results show, that the exchange of the original transcription initiation region by that of φ10 promoter from T7 and the insertion of the ccdAB locus in addition had no negative effect on plasmid maintenance. The elimination of one cer site producing pWA21 and pWA22 has led to a negligible raise of plasmid loss of 5–8% in approximately 50 generations, a faint disadvantage which is acceptable in regard to the enhanced expression. In contrast, the deletion of rop added more instability to the vectors, for instance 25% of the cells bearing pWA23 had lost their plasmids. Furthermore, the combination of Δrop and ccdAB increased the percentage of plasmid free cells to 32% (pWA24). This observation is not according to expectations, because the addiction module should mediate programmed cell death if the gene coding for the unstable antidote is lost. Moreover, the combination of one cer site and Δrop had a remarkably negative impact on plasmid persistence, as it resulted in a plasmid withdrawal of about 68%. Again, the addition of ccdAB adversely affected the plasmid stability. Apparently, combination of the specific genetic modules cer, rop and ccdAB, which are supposed to have a stabilizing impact on plasmids, does not simply lead to summable effects, but can contrarily destabilize vectors. In general, the influence on plasmid maintenance of plasmid-borne elements and their interaction with the particular host strain has to be examined in individual cases.


Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations.

Wegerer A, Sun T, Altenbuchner J - BMC Biotechnol. (2008)

Plasmid stability after 48 h in liquid culture without antibiotic selection. Cells were grown at 37°C in LB supplemented with 0.2% (w/v) L-rhamnose for 24 h, starting from this culture fresh medium was inoculated, and again incubated for 24 h under the same conditions, which roughly matches 50 generations. The percentage of cells without plasmid or loss of fluorescence are shown, the values are the averages of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254391&req=5

Figure 4: Plasmid stability after 48 h in liquid culture without antibiotic selection. Cells were grown at 37°C in LB supplemented with 0.2% (w/v) L-rhamnose for 24 h, starting from this culture fresh medium was inoculated, and again incubated for 24 h under the same conditions, which roughly matches 50 generations. The percentage of cells without plasmid or loss of fluorescence are shown, the values are the averages of three independent experiments.
Mentions: The usability of a plasmid is not only determined by the amount of protein produced in a specific combination of strain, gene and plasmid, but also by the stability of the expression vector. Especially if the induction is carried out on a large scale and during a longer period of process, plasmid loss can have a tremendous effect on total yield. In order to test the influence of the variations of the genetic modules, we determined how many cells lost their plasmids during a prolonged induction. If the cultures were induced at 30°C for 48 or 96 hours, which is roughly equivalent to 50 generations or 100 generations, respectively, no plasmid loss could be observed in none of the cultures (data not shown). But if the induction was carried out at 37°C for 48 hours, major differences between the plasmids could be detected. The originating pJOE4056.2 and its two direct derivatives pJOE5058.1 and pWA19 were perfectly stable (Fig. 4). These results show, that the exchange of the original transcription initiation region by that of φ10 promoter from T7 and the insertion of the ccdAB locus in addition had no negative effect on plasmid maintenance. The elimination of one cer site producing pWA21 and pWA22 has led to a negligible raise of plasmid loss of 5–8% in approximately 50 generations, a faint disadvantage which is acceptable in regard to the enhanced expression. In contrast, the deletion of rop added more instability to the vectors, for instance 25% of the cells bearing pWA23 had lost their plasmids. Furthermore, the combination of Δrop and ccdAB increased the percentage of plasmid free cells to 32% (pWA24). This observation is not according to expectations, because the addiction module should mediate programmed cell death if the gene coding for the unstable antidote is lost. Moreover, the combination of one cer site and Δrop had a remarkably negative impact on plasmid persistence, as it resulted in a plasmid withdrawal of about 68%. Again, the addition of ccdAB adversely affected the plasmid stability. Apparently, combination of the specific genetic modules cer, rop and ccdAB, which are supposed to have a stabilizing impact on plasmids, does not simply lead to summable effects, but can contrarily destabilize vectors. In general, the influence on plasmid maintenance of plasmid-borne elements and their interaction with the particular host strain has to be examined in individual cases.

Bottom Line: The introduction of a stem loop into the translation initiation region of the rhaPBAD promoter resulted in the most significant improvement of eGFP expression.The plasmid pWA21, containing the stem loop, one cer site and rop, attained high expression levels accompanied by a good stability, and on that score seems to be a well-balanced choice.The genetic modules showed a complex interplay, therefore two positive effects combined sometimes resulted in a disadvantage.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Industrielle Genetik, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. a_wegerer@yahoo.de

ABSTRACT

Background: A capable expression vector is mainly characterized by its production efficiency, stability and induction response. These features can be influenced by a variation of modifications and versatile genetic modules.

Results: We examined miscellaneous variations of a rhaPBAD expression vector. The introduction of a stem loop into the translation initiation region of the rhaPBAD promoter resulted in the most significant improvement of eGFP expression. Starting from this plasmid, we constructed a set of expression vectors bearing different genetic modules like rop, ccdAB, cer and combinations thereof, and tested the efficiency of expression and plasmid stability. The plasmid pWA21, containing the stem loop, one cer site and rop, attained high expression levels accompanied by a good stability, and on that score seems to be a well-balanced choice.

Conclusion: We report the generation of variations of the rhaPBAD expression vector and characterization hereof. The genetic modules showed a complex interplay, therefore two positive effects combined sometimes resulted in a disadvantage.

Show MeSH
Related in: MedlinePlus