TMAXTREE DREM cell successfully facilitates the high-throughput screening of antagonistic bacteria
In this issue, we recommend the work of Prof. Zhu Li's group in the School of Life Sciences, Guizhou University, related to high-throughput screening based on microfluidic technology. The group screened antagonistic bacteria through a droplet microfluidic platform, which improved the screening efficiency by about 3000 times compared with the traditional agar plate screening method. The mutants obtained from the screening showed a 62% increase in bacterial inhibitory activity compared to the wild-type strain.
Figure 1 High-throughput screening process of antagonistic bacteria
Soft rot is a bacterial plant disease that severely affects crops and is mainly caused by the soft-rotting bacterium Erwinia carotovora, which occurs in the fleshy storage tissues of vegetable and ornamental plants. This disease can spread rapidly within the plant, causing tissue hydrolysis, rotting, and ultimately resulting in reduced or even total crop loss, thus causing huge economic losses to agricultural production and becoming one of the most important factors limiting sustainable agricultural development.
Soft rot is a bacterial plant disease that severely affects crops and is mainly caused by the soft-rotting bacterium Erwinia carotovora, which occurs in the fleshy storage tissues of vegetable and ornamental plants. This disease can spread rapidly within the plant, causing tissue hydrolysis, rotting, and ultimately resulting in reduced or even total crop loss, thus causing huge economic losses to agricultural production and becoming one of the most important factors limiting sustainable agricultural development.
With the rapid development of modern biotechnology, biological control has been increasingly emphasized because of its good biological control effect, non-toxicity, harmlessness, non-pollution and other characteristics. Agricultural antibiotics (bioactive substances), antagonistic microorganisms and so on have been better applied in the research of pest control. The traditional screening of antagonistic bacteria is usually carried out by agar plate method, which has the limitations of high labor intensity, high cost and low efficiency, significantly affecting the screening and application of antagonistic bacteria. With the development of droplet microfluidic technology, its high-throughput characteristics were utilized to construct a new screening platform-DREM Cell, which significantly improves the screening efficiency while drastically reduces the cost.
Soil samples were collected from 10 cm below the surface of a konjac planting area in Bijie City, Guizhou Province, and the microbial resources were probed using the droplet system and the Petri dish system, respectively. The results of microscopic examination showed that the number of bacterial-containing droplets accounted for about 12.22% of the total number of droplets, and the number of colonies in droplets was basically the same as that of plates, indicating that the growth of strains in droplets was comparable to that of plate culture (Figure 2). Analysis of the colonies obtained under different culture conditions revealed that more than 95% of the OTUs in the droplet culture samples belonged to rare biotopes, and their relative abundance was less than 0.01% in the original soil samples (Fig. 3), which indicates that droplet culture has great potential for revealing rare biotopes in complex microbial communities.
Figure. 2 Microscope images of droplet generation and incubation of droplet samples
Figure 3 Comparison of amplicon sequencing results of different culture samples
Utilizing the GFP-Ecc15 strain as a reporter, the antagonist bacteria could inhibit the growth of the reporter strain, thus significantly reducing the fluorescence value of the system (Figs. 4, 5). The suitability of the screening system was verified in the enrichment screening of the mixed system of antagonist B. velezensis and non-antagonist E. coli, and the high-throughput screening model of antagonist bacteria was established. The maximum enrichment efficiency of the antagonist bacteria reached 226-fold, and the average enrichment efficiency of the antagonist bacteria in three replicate experiments increased 148-fold. The results demonstrated that the screening model based on droplet microfluidics could well isolate the bacteria with inhibitory effects on the reporter strains.
Figure 4 Growth of GFP-Ecc15 in droplets
Figure 5 Effect of antagonistic and non-antagonistic bacteria on the fluorescence value of GFP-Ecc15
Finally, based on this high-throughput model, screening of antagonistic strains was performed on complex samples of soil environment. Sorting was carried out at a rate of 105 cells per hour, and the sorted droplets were applied to the plates, and single colonies were selected to verify their inhibitory ability against the pathogen indicator bacterium Ecc15 by agar diffusion method. After screening, 32 bacterial strains with antimicrobial activity were enriched, among which the best strain achieved an inhibitory diameter of 20.86±1.56 mm. After ARTP mutagenesis, the inhibitory diameter was further enlarged to 26.15±0.29 mm, which was significantly larger than that of the starting strain of 18.31±0.64 mm (Figure 6), and the inhibitory activity was enhanced by 62%.
Figure 6 Differences in the circle of inhibition between the highly active mutant and the wild type
In this study, we combined droplet microfluidics with ARTP mutagenesis to carry out high-throughput antagonistic bacteria screening using the DREM Cell platform for environmental microbial resource mining, screening of antagonistic strains from complex samples, and screening of strain mutation libraries. Compared with the traditional method, the reagent consumption of culture medium was reduced to 1.2×107, and the screening rate was increased by more than 3,000 times, and highly efficient antagonistic bacteria were successfully screened and mutagenized from soil samples. This platform provides a more efficient and less costly solution for the screening of antagonistic bacteria, and offers a new perspective for agricultural biological control, which is important for the in-depth understanding and utilization of biological resources hidden in tiny soil particles.
Paper DOI: 10.27047/d.cnki.ggudu.2023.001995
TMAXTREE DREM cell successfully facilitates the high-throughput screening of antagonistic bacteria
In this issue, we recommend the work of Prof. Zhu Li's group in the School of Life Sciences, Guizhou University, related to high-throughput screening based on microfluidic technology. The group screened antagonistic bacteria through a droplet microfluidic platform, which improved the screening efficiency by about 3000 times compared with the traditional agar plate screening method. The mutants obtained from the screening showed a 62% increase in bacterial inhibitory activity compared to the wild-type strain.
Figure 1 High-throughput screening process of antagonistic bacteria
Soft rot is a bacterial plant disease that severely affects crops and is mainly caused by the soft-rotting bacterium Erwinia carotovora, which occurs in the fleshy storage tissues of vegetable and ornamental plants. This disease can spread rapidly within the plant, causing tissue hydrolysis, rotting, and ultimately resulting in reduced or even total crop loss, thus causing huge economic losses to agricultural production and becoming one of the most important factors limiting sustainable agricultural development.
Soft rot is a bacterial plant disease that severely affects crops and is mainly caused by the soft-rotting bacterium Erwinia carotovora, which occurs in the fleshy storage tissues of vegetable and ornamental plants. This disease can spread rapidly within the plant, causing tissue hydrolysis, rotting, and ultimately resulting in reduced or even total crop loss, thus causing huge economic losses to agricultural production and becoming one of the most important factors limiting sustainable agricultural development.
With the rapid development of modern biotechnology, biological control has been increasingly emphasized because of its good biological control effect, non-toxicity, harmlessness, non-pollution and other characteristics. Agricultural antibiotics (bioactive substances), antagonistic microorganisms and so on have been better applied in the research of pest control. The traditional screening of antagonistic bacteria is usually carried out by agar plate method, which has the limitations of high labor intensity, high cost and low efficiency, significantly affecting the screening and application of antagonistic bacteria. With the development of droplet microfluidic technology, its high-throughput characteristics were utilized to construct a new screening platform-DREM Cell, which significantly improves the screening efficiency while drastically reduces the cost.
Soil samples were collected from 10 cm below the surface of a konjac planting area in Bijie City, Guizhou Province, and the microbial resources were probed using the droplet system and the Petri dish system, respectively. The results of microscopic examination showed that the number of bacterial-containing droplets accounted for about 12.22% of the total number of droplets, and the number of colonies in droplets was basically the same as that of plates, indicating that the growth of strains in droplets was comparable to that of plate culture (Figure 2). Analysis of the colonies obtained under different culture conditions revealed that more than 95% of the OTUs in the droplet culture samples belonged to rare biotopes, and their relative abundance was less than 0.01% in the original soil samples (Fig. 3), which indicates that droplet culture has great potential for revealing rare biotopes in complex microbial communities.
Figure. 2 Microscope images of droplet generation and incubation of droplet samples
Figure 3 Comparison of amplicon sequencing results of different culture samples
Utilizing the GFP-Ecc15 strain as a reporter, the antagonist bacteria could inhibit the growth of the reporter strain, thus significantly reducing the fluorescence value of the system (Figs. 4, 5). The suitability of the screening system was verified in the enrichment screening of the mixed system of antagonist B. velezensis and non-antagonist E. coli, and the high-throughput screening model of antagonist bacteria was established. The maximum enrichment efficiency of the antagonist bacteria reached 226-fold, and the average enrichment efficiency of the antagonist bacteria in three replicate experiments increased 148-fold. The results demonstrated that the screening model based on droplet microfluidics could well isolate the bacteria with inhibitory effects on the reporter strains.
Figure 4 Growth of GFP-Ecc15 in droplets
Figure 5 Effect of antagonistic and non-antagonistic bacteria on the fluorescence value of GFP-Ecc15
Finally, based on this high-throughput model, screening of antagonistic strains was performed on complex samples of soil environment. Sorting was carried out at a rate of 105 cells per hour, and the sorted droplets were applied to the plates, and single colonies were selected to verify their inhibitory ability against the pathogen indicator bacterium Ecc15 by agar diffusion method. After screening, 32 bacterial strains with antimicrobial activity were enriched, among which the best strain achieved an inhibitory diameter of 20.86±1.56 mm. After ARTP mutagenesis, the inhibitory diameter was further enlarged to 26.15±0.29 mm, which was significantly larger than that of the starting strain of 18.31±0.64 mm (Figure 6), and the inhibitory activity was enhanced by 62%.
Figure 6 Differences in the circle of inhibition between the highly active mutant and the wild type
In this study, we combined droplet microfluidics with ARTP mutagenesis to carry out high-throughput antagonistic bacteria screening using the DREM Cell platform for environmental microbial resource mining, screening of antagonistic strains from complex samples, and screening of strain mutation libraries. Compared with the traditional method, the reagent consumption of culture medium was reduced to 1.2×107, and the screening rate was increased by more than 3,000 times, and highly efficient antagonistic bacteria were successfully screened and mutagenized from soil samples. This platform provides a more efficient and less costly solution for the screening of antagonistic bacteria, and offers a new perspective for agricultural biological control, which is important for the in-depth understanding and utilization of biological resources hidden in tiny soil particles.
Paper DOI: 10.27047/d.cnki.ggudu.2023.001995
