Nikolic, NelaISTA ; Sauert, Martina; Albanese, Tanino G.; Moll, Isabella
Objective: MazF is a sequence-specific endoribonuclease-toxin of the MazEF toxin–antitoxin system. MazF cleaves single-stranded ribonucleic acid (RNA) regions at adenine–cytosine–adenine (ACA) sequences in the bacterium Escherichia coli. The MazEF system has been used in various biotechnology and synthetic biology applications. In this study, we infer how ectopic mazF overexpression affects production of heterologous proteins. To this end, we quantified the levels of fluorescent proteins expressed in E. coli from reporters translated from the ACA-containing or ACA-less messenger RNAs (mRNAs). Additionally, we addressed the impact of the 5′-untranslated region of these reporter mRNAs under the same conditions by comparing expression from mRNAs that comprise (canonical mRNA) or lack this region (leaderless mRNA). Results: Flow cytometry analysis indicates that during mazF overexpression, fluorescent proteins are translated from the canonical as well as leaderless mRNAs. Our analysis further indicates that longer mazF overexpression generally increases the concentration of fluorescent proteins translated from ACA-less mRNAs, however it also substantially increases bacterial population heterogeneity. Finally, our results suggest that the strength and duration of mazF overexpression should be optimized for each experimental setup, to maximize the heterologous protein production and minimize the amount of phenotypic heterogeneity in bacterial populations, which is unfavorable in biotechnological processes.
BMC Research Notes
We acknowledge the Max Perutz Labs FACS Facility together with Thomas Sauer. NN is grateful to Călin C. Guet for his support. This work was funded by the Elise Richter grant V738 of the Austrian Science Fund (FWF), and the FWF Lise Meitner grant M1697, to NN; and by the FWF grant P22249, FWF Special Research Program RNA-REG F43 (subproject F4316), and FWF doctoral program RNA Biology (W1207), to IM. Open access funding provided by the Austrian Science Fund.
Nikolic N, Sauert M, Albanese TG, Moll I. Quantifying heterologous gene expression during ectopic MazF production in Escherichia coli. BMC Research Notes. 2022;15. doi:10.1186/s13104-022-06061-9
Nikolic, N., Sauert, M., Albanese, T. G., & Moll, I. (2022). Quantifying heterologous gene expression during ectopic MazF production in Escherichia coli. BMC Research Notes. Springer Nature. https://doi.org/10.1186/s13104-022-06061-9
Nikolic, Nela, Martina Sauert, Tanino G. Albanese, and Isabella Moll. “Quantifying Heterologous Gene Expression during Ectopic MazF Production in Escherichia Coli.” BMC Research Notes. Springer Nature, 2022. https://doi.org/10.1186/s13104-022-06061-9.
N. Nikolic, M. Sauert, T. G. Albanese, and I. Moll, “Quantifying heterologous gene expression during ectopic MazF production in Escherichia coli,” BMC Research Notes, vol. 15. Springer Nature, 2022.
Nikolic N, Sauert M, Albanese TG, Moll I. 2022. Quantifying heterologous gene expression during ectopic MazF production in Escherichia coli. BMC Research Notes. 15, 173.
Nikolic, Nela, et al. “Quantifying Heterologous Gene Expression during Ectopic MazF Production in Escherichia Coli.” BMC Research Notes, vol. 15, 173, Springer Nature, 2022, doi:10.1186/s13104-022-06061-9.
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