Optimization and Comparison of Different Methods and Factors for Efficient Transformation of Brucella abortus RB51strain

Abstract

Background: The development of protective vaccines for Brucella spp. has been hampered by the difficulty in transformation of Brucella cells with foreign DNA for genetic manipulation. It seems that the formation of Brucella spheroplasts would increase the efficiency of transformation. The aim of this study was to devise an efficient method for the transformation of Brucella spp. Materials and Methods: At first, spheroplast of Brucella was prepared by glycine and ampicillin induction and transformed using optimized protocols of CaCl 2 , electroporation, and lipofection methods. Then, the efficacy of transformation was compared between the three-mentioned methods. Results: Ampicillin-induced spheroplasts from early-log phase culture of brucella when incubated in a medium-containing 0.2 M sucrose during cell recovery had higher transformation efficiency in three different methods. Comparison of the transformation efficiency of Brucella abortus RB51 using the CaCl 2 , lipofection, and electroporation methods revealed that the transformation efficiency with the lipofection method was significantly higher than with other two methods ( P < 0.05). Conclusions: Lipofection method by lipofectamine 2000 on ampicillin-induced spheroplasts can be a suitable approach for Brucella transformation.

Engineering the Maize Root Microbiome: A Rapid MoClo Toolkit and Identification of Potential Bacterial Chassis for Studying Plant–Microbe Interactions

“…Transformation efficiency varies drastically between microbes and based on the transformation method employed; thus, optimization of a DNA transformation protocol is typically developed separately for each strain. − To streamline experimentation within SynCom, we aimed to develop a standardized transformation protocol that works for each strain of SynCom to enable high-throughput experimentation. Electroporation was chosen over other methods of DNA transformation as it has offered the highest transformation efficiency for the majority of SynCom relatives in prior work. − , We first tested different electroporation voltages for each strain (Figure A). The strains Bpi and Hro had clear optimal voltages of 3.0 and 1.2 kV, respectively, with the rest of the strains having a larger range of voltages being optimal.…”

“…Electroporation was chosen over other methods of DNA transformation as it has offered the highest transformation efficiency for the majority of SynCom relatives in prior work. [54][55][56]63 We first tested different electroporation voltages for each strain (Figure 2A). The strains Bpi and Hro had clear optimal voltages of 3.0 and 1.2 kV, respectively, with the rest of the strains having a larger range of voltages being optimal.…”

“…Transposon mutants have been generated in Herbaspirillum , and Stenotrophomonas species, , expression vectors have been constructed for Brucella species, , but no tools are available for Chryseobacterium and Curtobacterium species to the author’s knowledge. Furthermore, transformation methods have yet to be established for Chryseobacterium species, but either electroporation and/or conjugation has been used to transform species in the same genera for the rest of the SynCom. − …”

Design and Characterization of a Recombinant Brucella abortus RB51 Vaccine That Elicits Enhanced T Cell-Mediated Immune Response

“…Vaccines 2022, 10, 388. https://doi.org/10.3390/vaccines10030388 https://www.mdpi.com/journal/vaccines economic losses in the world livestock sector and undulant fever in humans [1,2]. Cattle are a typical host for Brucella abortus, although Brucella abortus can cause infections in sheep, goats, pigs, bison, buffalo, horses, elk, and many other animal species.…”

“…The genus Brucella comprises Gram-negative intracellular bacterial pathogens that cause brucellosis, resulting in abortion in domestic animals and causing considerable economic losses in the world livestock sector and undulant fever in humans [ 1 , 2 ]. Cattle are a typical host for Brucella abortus , although Brucella abortus can cause infections in sheep, goats, pigs, bison, buffalo, horses, elk, and many other animal species.…”