The study on the production of antibiotics in streptomyces and biosynthesis is often disturbed due to the variations observed in successor and parallel cultivation of a batch of the same kind of strain in the Erlenmeyer flask. These flasks have a baffle or sometimes a stainless-steel spring. They both help in increasing the aeration and thus reducing the cell aggregates’ size. Now, this is what leads to the splashing of liquid which helps in cell wall growth. It probably leads to the variation in results. There is a high standard deviation in production rates due to which parallel cultures should be increased. What this will do is distinguish the critical differences from variations. An increase in parallel cultures would also demand increased resources such as flasks, shakers, analytical equipment, and etc.
However, if there is a cultivation method with more minor variations in secondary metabolite production, the cost of resources and workload will be reduced.
Now, 24-square deep well plates have come into the study for systems biology research in Streptomyces coelicolor. The current study that you will read below was established for the need for a production system with low variability. This is for the investigation required on regulatory genes of aminocoumarin antibiotic formation in the heterologous producer strain.
The primary materials that are utilized in this study are mentioned below:
- Bacterial strains
Cultivation in Erlenmeyer flask
The selection of recombinant strains was made with thiostrepton and kanamycin. As usual, the S. coelicolor M512 was put into a 300 ml Erlenmeyer flask. This flask had a stainless-steel spring. For pre-cultivation, 50ml YMG medium with 1% malt extract, 0.4% yeast extract, and 0.4% glucose with 12.5 μg ml− 1 was used. The pre-cultivation was done at 200rpm in 30 degrees Celsius for two days.
After this, for Streptomyces tendae Tü901/8c cultivation, 50 μl spore suspension was incubated in a medium of 500 μl 2xYT. This incubation was carried out for 10 minutes at 55 degrees Celsius. Once incubation was done, cultivation was done at 30 degrees Celsius for 2 hours at 200 rpm.
Cultivation in a 24-square deep well plate
The S. coelicolor M512(novBG01) was prepared with YGM exactly as cultivated in the Erlenmeyer flask. After preparation of preculture, 0.5 ml of it was mixed with CDM measuring 40ml. Once this mixture was prepared, 3 ml of it was placed into each deep well plate. The deep well plate was then left for cultivation at 30 degrees Celsius for 7 days at 300 rpm.
- tendae Tü901/8c spores were also germinated and pre-cultured as described above. After this, they were used to inoculate a 50ml production medium. This mixture was then placed in deep well plates with a volume of 3ml in each well.
Homogenized and frozen inoculum preparation
The preparation of both these mediums followed the procedure given by Bapat et al., but do note its unpublished data.
The fresh, homogenized inoculum was prepared by taking 50ml of YGM. This preculture was then centrifuged at 2800×g and 4-8 degrees Celsius for 10 minutes. After this, the cells that were prepared were homogenized carefully with a Potter homogenizer. It is operated manually. The mixture prepared through this procedure is the one used for inoculation.
The frozen inoculum was prepared with cells prepared through the centrifugation of YGM pre cultures. The preculture was resuspended in 1/5 of the original culture volume. The original culture volume is an aqueous solution of 20% (w/v) peptone. It was then homogenized with the same method as in the preparation of homogenized inoculum. The resulting mixture was then divided into aliquots and stored at a shallow temperature of -70 degrees Celsius.
After this, specific other method and preparations were carried out that are all listed below:
- Addition of siloxylated ethylene oxide/propylene oxide copolymer
- Determination of dry cell weight
- Determination of optical density at 600 nm (OD600)
- Analysis of secondary metabolites
In this experiment, the Erlenmeyer flasks and deep well plates are compared through the novobiocin production of S. coelicolor M512(novBG01). It was concluded that production after 7 days was higher in the Erlenmeyer flask. Still, it was visible the variability was much lower in deep well plates, which was the required result as well.
Stefanie Siebenberg, P. M. (2010). Reducing the variability of antibiotic production in Streptomyces by cultivation in 24-square deepwell plates. Journal of Bioscience and Bioengineering , 230-234.