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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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Effect of plasmids, carbohydrates and eGFP expression on cell length of different strains. (A) The strains were grown for 24 h at 30°C in LB supplemented with 0.2% (w/v) L-rhamnose (+), L-arabinose (A) or D-glucose (G) as indicated in the row 'induction'. For each culture the lengths of 100 cells were determined, the average value and the standard deviation are shown. The optical density of the cultures is indicated by a black diamond above the bar. The number of cells per milliliter of culture were determined by counting them in a Thoma-chamber.
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Figure 6: Effect of plasmids, carbohydrates and eGFP expression on cell length of different strains. (A) The strains were grown for 24 h at 30°C in LB supplemented with 0.2% (w/v) L-rhamnose (+), L-arabinose (A) or D-glucose (G) as indicated in the row 'induction'. For each culture the lengths of 100 cells were determined, the average value and the standard deviation are shown. The optical density of the cultures is indicated by a black diamond above the bar. The number of cells per milliliter of culture were determined by counting them in a Thoma-chamber.

Mentions: When induction of eGFP was carried out in the E. coli strains BW3110 and BL21 Rha- additionally to the experiments in JM109 mentioned above, lower values of fluorescence were observed (referring to 100 μl of a cell suspension of 0.1 OD600, data not shown). However, the relations between the fluorescence obtained with the individual vectors were comparable. But we noticed that in BW3110 and BL21 Rha- the optical density of the cultures increased significantly, though these strains are not able to use L-rhamnose as a carbon source, due to the lack of rhaB. Examination by microscopy showed, that the reason for this raise was not a higher cell count, but an increased cell length (Fig. 5). As these differences were most significant in BL21 Rha-, this strain was used to examine the effect on morphology in more detail (Fig. 6).


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)

Effect of plasmids, carbohydrates and eGFP expression on cell length of different strains. (A) The strains were grown for 24 h at 30°C in LB supplemented with 0.2% (w/v) L-rhamnose (+), L-arabinose (A) or D-glucose (G) as indicated in the row 'induction'. For each culture the lengths of 100 cells were determined, the average value and the standard deviation are shown. The optical density of the cultures is indicated by a black diamond above the bar. The number of cells per milliliter of culture were determined by counting them in a Thoma-chamber.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Effect of plasmids, carbohydrates and eGFP expression on cell length of different strains. (A) The strains were grown for 24 h at 30°C in LB supplemented with 0.2% (w/v) L-rhamnose (+), L-arabinose (A) or D-glucose (G) as indicated in the row 'induction'. For each culture the lengths of 100 cells were determined, the average value and the standard deviation are shown. The optical density of the cultures is indicated by a black diamond above the bar. The number of cells per milliliter of culture were determined by counting them in a Thoma-chamber.
Mentions: When induction of eGFP was carried out in the E. coli strains BW3110 and BL21 Rha- additionally to the experiments in JM109 mentioned above, lower values of fluorescence were observed (referring to 100 μl of a cell suspension of 0.1 OD600, data not shown). However, the relations between the fluorescence obtained with the individual vectors were comparable. But we noticed that in BW3110 and BL21 Rha- the optical density of the cultures increased significantly, though these strains are not able to use L-rhamnose as a carbon source, due to the lack of rhaB. Examination by microscopy showed, that the reason for this raise was not a higher cell count, but an increased cell length (Fig. 5). As these differences were most significant in BL21 Rha-, this strain was used to examine the effect on morphology in more detail (Fig. 6).

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