Development of oriC-Based Plasmids for Mesoplasma florum

Area of Science:

• Synthetic Biology
• Microbiology
• Systems Biology

Background:

• Mesoplasma florum is a near-minimal bacterium with potential for systems biology and synthetic biology applications.
• Limited genetic engineering tools have hindered understanding and modification of M. florum's genome.
• M. florum's rapid growth, non-pathogenic nature, and small genome make it an ideal chassis.

Purpose of the Study:

• To develop the first artificial plasmids capable of replication in M. florum.
• To establish efficient transformation methods for M. florum.
• To enable genetic engineering of M. florum for synthetic biology applications.

Main Methods:

• Evaluation of M. florum's susceptibility to common antibiotics.
• Construction and testing of artificial plasmids using M. florum's origin of replication (oriC) regions.
• Development of polyethylene glycol-mediated transformation, electroporation, and conjugation methods.

Main Results:

• Plasmids containing specific M. florum oriC regions (rpmH-dnaA and dnaA-dnaN) achieved transformation frequencies of ~4.1 × 10^-6 and were stably maintained.
• Alternative transformation methods (electroporation, conjugation) yielded frequencies up to 7.87 × 10^-6 and 8.44 × 10^-7 transformants per viable cell.
• Functional antibiotic resistance genes (tetracycline, puromycin, spectinomycin/streptomycin) were demonstrated in M. florum.

Conclusions:

• The developed artificial plasmids and transformation methods are the first for M. florum.
• These genetic tools are crucial for advancing M. florum as a minimal cellular chassis in synthetic biology.
• This work provides a strong foundation for future genome engineering efforts in M. florum.