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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

Schematical overview of the vectors used in this study. A physical map for relevant restriction endonucleases is given for the plasmid pJOE4056.1 and the location and orientation of the rhaPBAD promotor, the genes encoding eGFP, ampicillin resistance (bla), rop and addiction modules ccdA and ccdB are indicated by triangle and arrows. The transcription terminator sequence (ter) is derived from the E. coli rrnB operon.
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Figure 1: Schematical overview of the vectors used in this study. A physical map for relevant restriction endonucleases is given for the plasmid pJOE4056.1 and the location and orientation of the rhaPBAD promotor, the genes encoding eGFP, ampicillin resistance (bla), rop and addiction modules ccdA and ccdB are indicated by triangle and arrows. The transcription terminator sequence (ter) is derived from the E. coli rrnB operon.

Mentions: In this study we tested the influence of a modification of the transcription initiation region, the presence of three genetic modules on the expression vectors and combinations thereof. The L-rhamnose-inducible vector pJOE4056.1 had already included the first 19 base pairs (bp) upstream of the AUG start codon from the highly translated bacteriophage T7 gene 10. This sequence included a SD sequence perfectly matching the E. coli canonical sequence but lacked the stem-loop structure. In the first step, the original transcription initiation region of rhaPBAD on pJOE4056.2 was replaced by a modified T7 gene 10 untranslated leader sequence which included the stem-loop structure. The difference to the original T7 gene 10 upstream sequence [26] is a deletion of an 8 bp sequence between the stem-loop and SD which was replaced by the recognition site of restriction endonuclease AflII. We have named the resulting promotor rhaPBAD-T7sl (stem-loop) and the corresponding plasmid pJOE5058.1, which was the source of the subsequent modifications. In the next step, a ccdAB cassette was inserted (pWA19), one of the two cer sites was excised (pWA21) and the rop site was deleted (pWA23). Plasmids with all possible combinations of these modifications were constructed (Tab. 1 and Fig. 1).


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)

Schematical overview of the vectors used in this study. A physical map for relevant restriction endonucleases is given for the plasmid pJOE4056.1 and the location and orientation of the rhaPBAD promotor, the genes encoding eGFP, ampicillin resistance (bla), rop and addiction modules ccdA and ccdB are indicated by triangle and arrows. The transcription terminator sequence (ter) is derived from the E. coli rrnB operon.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematical overview of the vectors used in this study. A physical map for relevant restriction endonucleases is given for the plasmid pJOE4056.1 and the location and orientation of the rhaPBAD promotor, the genes encoding eGFP, ampicillin resistance (bla), rop and addiction modules ccdA and ccdB are indicated by triangle and arrows. The transcription terminator sequence (ter) is derived from the E. coli rrnB operon.
Mentions: In this study we tested the influence of a modification of the transcription initiation region, the presence of three genetic modules on the expression vectors and combinations thereof. The L-rhamnose-inducible vector pJOE4056.1 had already included the first 19 base pairs (bp) upstream of the AUG start codon from the highly translated bacteriophage T7 gene 10. This sequence included a SD sequence perfectly matching the E. coli canonical sequence but lacked the stem-loop structure. In the first step, the original transcription initiation region of rhaPBAD on pJOE4056.2 was replaced by a modified T7 gene 10 untranslated leader sequence which included the stem-loop structure. The difference to the original T7 gene 10 upstream sequence [26] is a deletion of an 8 bp sequence between the stem-loop and SD which was replaced by the recognition site of restriction endonuclease AflII. We have named the resulting promotor rhaPBAD-T7sl (stem-loop) and the corresponding plasmid pJOE5058.1, which was the source of the subsequent modifications. In the next step, a ccdAB cassette was inserted (pWA19), one of the two cer sites was excised (pWA21) and the rop site was deleted (pWA23). Plasmids with all possible combinations of these modifications were constructed (Tab. 1 and Fig. 1).

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