Isolation of Antibiotic-Producing Cells from the Objects in Agar Cultures of Synthetic DNA (Streptomyces) Crown Cells with Monolaurin and Egg White

Artificial cells that are fully functional (self-replicating) were first reported in 2012 [1], and the principal methods to prepare such artificial cells were reported in 2016 [2]. In 2016, artificial cells with an outer membrane covered in DNA were referred to as DNA crown cells [3]. The DNA crown cells were synthesized using four common commercial compounds (sphingosine (Sph), DNA, adenosine, and monolaurin) and could be cultured in egg white.

In previous studies, cell proliferation and the appearance of various types of objects were observed under a microscope when monolaurin-treated synthetic DNA (Escherichia coli, human placenta, Ascidian, or Streptomyces) crown cells were cultivated with or without egg white on agar plates [4-7].

It has also been demonstrated that antibiotic-producing cells could be isolated from beer produced using co-cultures of DNA crown cells and yeast stimulated with egg white or monolaurin, suggesting that egg white and/or monolaurin can induce the production of antibiotic producing cells [8]. In the present study, I examined whether antibiotic-producing cells could be isolated from the objects that form on agar plate cultures of synthetic DNA (Streptomyces) crown cells with monolaurin and/or egg white. Here, I describe the isolation of the antibiotic-producing cells, which I called antibiotic crown-Strept cells-c, from the objects.

Materials

The materials used were the same as those employed in previous studies [9,10], Sph (Tokyo Kasei, Japan), DNA (from Streptomyces), adenosine (Sigma-Aldrich, USA and Wako, Japan), monolaurin (Tokyo Kasei), and adenosine-monolaurin (A-M), a compound synthesized from mixing adenosine and monolaurin [9,10]. Monolaurin solutions were prepared to a final concentration of 0.1M in distilled water. Agar plates were prepared using standard agar medium (AS ONE, Japan). Dulbecco’s minimal Essential Eagle’s Medium (DMEM) (Sigma, USA), and bovine serum (Sigma, USA) were also used.

The materials used for testing the production of antibiotics were as follows. Malt (Black Rock, New Zealand) obtained from Advanced Brewing (Tokyo, Japan) and dry ale yeast (Safale S-04; Fermentis Bergy) from a handmade beer kit (Auvelcraft, Okazaki, Japan) were used according to the manufacturers’ instructions. Potato dextrose agar (Kyodo Nyugiou, Tokyo, Japan), Bacillus subtilis natto (Daikokuya, Nagoya, Japan) was also purchased.

Methods

Preparation of Synthetic DNA (Streptomyces) crown cells: The synthetic DNA (Streptomyces) crown cells were prepared as follows [9,10]. First, 180μL of Sph (10mM) and 50μL of DNA (1.7μg/μL) were combined, and the mixture was heated and cooled twice Next, A-M solution (100μL) was added, and the mixture was incubated at 37 °C for 15 min. Subsequently, 30μL of monolaurin solution was added, and the mixture was incubated at 37 °C for 5 min. The resulting suspension was used as the synthetic DNA (Streptomyces) crown cells.

Culturing of DNA (Streptomyces) crown cells with monolaurin and egg white: The culturing of DNA (Streptomyces) crown cells with monolaurin on agar plates was performed as follows. First, 50.0μL of sample was plated with a bacteria spreader on three agar plates (P-4, P-5, and P-6) that were 8.0cm in diameter. Then, 1.5mL of twice-diluted 0.1M monolaurin was immediately poured onto each agar plate. The excess monolaurin was removed and the plates were inverted and incubated at 37 °C for 5h. Subsequently, approximately 3.0mL of egg white was plated, the excess egg white was removed, and the plates were inverted and incubated at 37 °C for 7 days. Afterwards, the plates were stored at 4 °C

Collection and cultivation of the objects: The objects that had formed on P-4, P-5, and P-6 were collected as follows. First, approximately 6mL of distilled water was poured onto each of the plates. The water on the surface of the agar was swirled around in the dish to suspend the objects in the water, then the water was recovered (approximately 4mL/sample). The collected objects were cultivated on agar plates (8.0cm in diameter) or in liquid medium in tubes as follows.

For cultivation on agar plates, approximately 4mL of sample was mixed with an approximately equal volume of egg white.

Three milliliters of each mixture was poured onto separate plates and spread over the whole surface of the plate. Excess sample was removed, then the plates were inverted and incubated at 37 °C for 24h. Subsequently, the cultured plates were stored at 4 °C.

For cultivation in liquid medium, approximately 3mL of each mixture of egg white and sample was added to 30mL of DMEM containing 10% bovine serum, then incubated at 37 °C for 3 days in tubes. Subsequently, the cultured tubes were stored at 4 °C. These cultivated cells were used for the malt cultures.

Antibiotic production assay: For the antibiotic production assay, samples were prepared as follows. First, 0.5mL of the cell cultures in DMEM containing 10% bovine serum was added to 5.0mL of malt, and incubated for 7 days at room temperature (approximately 20 °C). Samples of cell culture liquid were collected and tested in the assay. The antibiotic production assay was carried out using the agar well method, as described previously [8]. Briefly, the test bacteria were mixed with 200mL of agar medium and dispensed into Petri plates. A well of approximately 2cm in diameter was made in the agar in each plate. The test fluid (approximately 400μL) was dispensed into the well in each plate, and the plates were incubated at 37 °C for 18h. After incubation, the presence of a zone of inhibition was observed.

Cell separation from antibiotic contained malt: Samples (20μL) of the malt culture liquids were dispensed into agar plates, and the plates were incubated at 37 °C for 18h.

Observation of the plates: Objects on plates were observed directly with the naked eye.

Figure 1 shows a photograph of an agar plate (P- 4) containing synthetic DNA (Streptomyces) crown cells after 7 days of culture with monolaurin and egg white. Circular objects of various sizes that were visible to the naked eye were observed over the whole Petri dish.

Figure 1:A photograph of an agar plate (plate no. 4) containing synthetic DNA (Streptomycess) crown cells after 7 days of culture with monolaurin and egg white. Circular objects of various sizes that can be seen by the naked eye are observed over the whole Petri dish, which has a diameter of 8.0cm.

Figure 2 shows a photograph of an agar plate containing the objects collected from P-4 and cultured for 24h; Many round objects similar to cell colonies could be seen by the naked eye on the Petri dish.

Figure 2:A photograph of an agar plate containing the objects collected from P-4 and cultured for 24h. Many round objects similar to cell colonies can be seen by the naked eye on the Petri dish, which has a diameter of 8.0cm.

Figure 3 shows a photograph of the liquid medium containing the objects collected from P-4 and cultured for 3 days. A change in the liquid color was observed when compared to the control liquid medium, suggesting that the objects were metabolized.

Figure 3:A photograph of the liquid medium containing the objects collected from P-4 and cultured for 3 days (right panel). A change in the liquid color is observed when compared to the control liquid medium (Light), suggesting that the objects were metabolized.

Figures 4, 5 and 6 show photographs of plates no. 4, 5 and 6 in the antibiotic production assay; a clear zone was observed on all plates.

Figure 4:A photograph of agar plate no. 4 in the antibiotic production assay. A clear zone is observed..

Figure 5:A photograph of agar plate no. 5 in the antibiotic production assay. A clear zone is observed.

Figure 6:A photograph of agar plate no. 6 in the antibiotic production assay. A clear zone is observed

Figure 7 shows a photograph of an agar plate containing the cell culture liquid of sample no. 6 prepared with malt for the antibiotic production assay and incubated at 37 °C for 18h; the growth of bacteria-like colonies was observed.

Figure 7:A photograph of an agar plate containing the cell culture liquid of sample no. 6 prepared with malt for the antibiotic production assay and incubated at 37 oC for 18h. The growth of bacteria-like colonies is observed.

Figure 8 shows a photograph of agar plate no. 6 that was prepared using the collected objects from sample no. 6 cultured with malt for the antibiotic production assay; a clear zone was observed.

Figure 8:A photograph of agar plate no. 6 that was prepared using the collected objects from sample no. 6 cultured with malt for the antibiotic production assay. A clear zone is observed.

Previously, I demonstrated that antibiotic was produced in the co-cultures of DNA (Streptomyces) crown cells and yeast used in beer production [11-14] and that the antibiotic producing cells were isolated from the beer [8]. It has been reported that various objects form in the agar cultures of synthetic DNA crown cells with egg white and monolaurin [4-7]. In the present study, I examined whether the antibiotic-producing cells could be isolated from these objects.

When the objects visible to the naked eye were collected and cultured on agar plates, the growth of colonies was observed on all plates. When the objects were cultivated in liquid medium and the medium was subsequently cultivated in malt medium, antibiotic was found in the malt cultures. Moreover, when the malt medium containing the antibiotic was cultivated on agar plates, colonies formed. Subsequently, cells from the colonies were cultivated with malt and without yeast, and antibiotic production was observed in the malt medium. These results demonstrated that antibiotic-producing cells were present in the objects that formed in the cultures of synthetic DNA crown cells with egg white and monolaurin. However, it remains unclear whether these cells and/or the antibiotic were the same as those that were produced and isolated from the co-cultures of DNA (Streptomyces) crown cells and yeast [8]. Also, as described in a previous paper [8], there have not yet been any studies on the origins of the cells in the objects, and there may be possibilities which were selected in nonscience, Nonetheless, the present experiments demonstrated that the samples stimulated with egg white and monolaurin contained antibiotic-producing cells that could be isolated.

If the cells were separated based on the scientific mechanism, it would be very important for the field of life sciences to clarify the origin of these cells. However, before such investigations, I believe that I should first clarify whether multiple types of antibioticproducing cells could be separated from the cultures with egg white or monolaurin.

As described in my previous paper [8], the antibiotic-producing DNA (Streptomyces) crown cells were named “antibiotic crown- Strep cells-c, with “c” representing “culture plates”. It is possible that multiple antibiotics were produced, and the antibiotic produced by the crown cells was called “crown antibiotic”. In conclusion, in our previous study, I found that antibiotic was produced in the cocultures of DNA (Streptomyces) crown cells and yeast, and in the present study, I was able to isolate the antibiotic-producing cells; the produced antibiotic in this study was named “crown antibiotic-2”.

The antibiotic-producing cells (antibiotic crown-Strep cells-c) could be isolated from the objects that form on agar plate cultures of synthetic DNA (Streptomyces) crown cells with monolaurin and egg white, The produced antibiotic in this study was named “crown antibiotic-2”.

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