Introduction of new antibiotics by screening antibacterial circles

Discover new antibiotics by screening the inhibition zone

In order to meet the growing demand for drug-resistant microbial therapy, a competitive evolution-based adaptive evolution screening method was developed. One of the challenges of this approach is how to find high-value strains from a diverse library of microbial clones and screen for the best candidate strains for downstream drug development that produce novel antibiotics.

For screening antibiotic-producing strains from clone libraries, agar plate inhibition zone or transparent circle screening is commonly used. Resistant clones can produce and secrete metabolites that inhibit plate microbes, thus creating a transparent circle around the clones that can be accurately screened from the clone pool based on the size of the transparent circle. The diameter of the transparent ring is generally proportional to the yield of resistant metabolites. Therefore, high value strains usually produce the largest diameter transparent circle. These clones were screened for further characterization studies and drug development.

If you use manual screening and pick clones that produce a zone of inhibition, the whole process is slow, cumbersome, and error-prone. To address these issues, Molecular Devices has developed the QPix400 family of microbial cloning screening systems that provide a high-throughput automated cloning screening and picking platform with unparalleled performance and efficiency. Here we describe in detail how to use this system and software modules to objectively screen and pick high-value transparent circle-producing strains.

Advantage

·Applicable to various adaptive evolution applications

· Speed ​​up the development of treatment methods for new drug-resistant strains

· User-defined software parameters to discover unique biological properties

· Screen high-value clones with reliable automation solutions

Identification of clones producing inhibition zones

Streptomyces clavuligerus can produce more than 20 biologically active secondary metabolites. Including many B-lactam antibiotics, such as cephamycin C, cephalosporin C, clavulanic acid and the like. Streptomyces clavuligerus (ATCC 27064) was used here as a model organism to demonstrate the identification and selection of the inhibition zone for E. coli (Migula, Castellani and Chalmers ATCC 47076). Both strains were obtained from ATCC and cultured using TSB medium. The liquid culture stage was incubated for 1-3 days using a shaker, and the corresponding S. clavuligerus was incubated at 37 ° C at 30 ° C and E. coli. After the culture of S. clavuligerus, the OD600 value of the diluted culture solution is between 0.10 and 0.13, and 2 ul of the culture solution is taken to the TSA agar plate, and cultured at 28 ° C for 3-5 days. After the growth of the S. clavuligerus TSA plate, the diluted E. coli broth was evenly spread onto the plate, ensuring that E. coli completely surrounded and contacted each S. clavuligerus clone. The plate was incubated at 28 ° C for 1 - 2 days and observed daily. As shown in Figure 1, the S. clavuligerus clone produced a zone of inhibition against E. coli.

The plate was imaged white using the QPix400 system's zone of inhibition identification software. As shown in Figure 2, the software identified clones with transparent circles. Advanced software algorithms allow users to customize screening parameters, select and select inhibitor zones that meet specific requirements, such as clone size, zone of inhibition, clone diameter, axial ratio, and clone edge shape (Figure 2).

Next, the software-selected clones that produce the zone of inhibition are arranged in thumbnail form. Clones can be arranged in a variety of orders, such as total transparent area size, clone size, axial ratio, edge shape, etc. (Figure 3). The software automatically lists detailed quantitative data for each clone that produces a zone of inhibition, making it easy to view at any time.

Screening and picking clones that produce inhibition zones based on clone libraries

In order to further verify the effect of the automatic inhibition zone recognition and screening method based on clone library or adaptive evolution, QTrays culture plates with various microbial clone libraries were screened. The dedicated software module accurately screened three clones that produced a zone of inhibition from the 48 clones grown on QTray (Figure 4). This approach enables high-throughput, purposeful screening, identification, and picking of clones that produce transparent circles from a large library of microbial clones.

to sum up

The market needs to continuously develop new antibiotics for the treatment of diseases caused by drug-resistant infections. In order to develop new antibiotics or fine-tune the biosynthetic pathway of antibiotics, researchers at home and abroad have made a lot of efforts. Both broad-based antibiotic overproduction strains can be generated based on clonal library screening or based on competitive adaptive evolution.

Using the QPix400 series of microbial cloning screening systems and the QPix inhibition zone software identification module, we can easily and reliably perform high-throughput screening of clear circles for cloning. This automated screening method will greatly increase our ability to find new antibiotics for the disease caused by existing or emerging antibiotic-resistant bacteria.