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light-dependent controlled lysis system

In order to be biologically safe, bacterial lysis is essential. This part can be easily used to achieve the lysis of bacteria under light conditions. In this system, under dark conditions, phosphorylated FixJ protein was transferred from YF1 protein to FixJ protein, and phosphorylated FixJ protein activated pFIXK2 promoter, then activated downstream gene expression.When the LacI protein binds to LacO, it prevents the downward expression of Lac promoter, the lysis gene is not activated and the cells grow normally. When cultured under light conditions,phosphorylation of FixJ protein was blocked and gene expression regulated by pfixk2 promoter was inhibited. Lac promoter expression was not inhibited, lysis gene expression was normal,then we achieve the lysis of bacteria. When using this system,you just need introduce the plasmid to the host cell.Then you could achieve the lysis of bacteria after you turn on the light.

Usage and Biology

In 2017, we established a light-controlled system that the bacteria can lysis in dark conditions. However, we faced a series of problems after that. For example, it is difficult for us to control the wavelength and intensity of light when culturing bacteria, because we have to keep the wavelength and intensity of light unchanged. So, we modify the system to achieve the cultivate of bacteria under dark condition. We use lactose operon and negative induction system and light-controlled system to establish a repression device.
In this system, under dark conditions, phosphate group of YF1 protein will be transferred to FixJ protein, and the phosphorylated FixJ protein will initiate the activation of FIXK2 promoter to drive the expression of downstream gene including eGFP and LacI. When the LacI protein was expressed, it will prevent the function of Lac promoter. As a result, the lysis gene will not be expressed, bacteria grow normally. When induced by light, phosphorylation of FixJ protein was blocked and gene expression regulated by pfixk2 promoter was inhibited. Lac promoter expression was not inhibited, lysis gene was expressed and cell lysed.

Characterize

After the recombinant plasmid was transferred into the host bacteria, the light-dependent controlled lysis system was established successfully. The corresponding bacteria was preserved by 30% after cultured for 12 hours. We conducted the following two experiments to verify the effectiveness of the light-dependent controlled lysis system.
(1) Pick single colony into 5 mL fresh culture medium. During 14 hours of culturing in dark and light conditions, we plotted the corresponding growth curve (Fig. 1).
(2) Single colonies were inoculated into two shaking flasks which containing ampicillin resistance. One shaking flask was cultured under light conditions while another one was cultured under dark conditions at 37 ℃ for 30 hours. Then, we used the Enzyme Labeling Instrument to measure the OD600. We obtained the following results (Fig. 2 and Fig. 3).

Experimental results

(1) It can be obviously seen that the test tube cultured under light conditions was much clearer than that cultured under dark conditions, which meant our light-controlled system was effective. We did three repeated experiments and drew the corresponding growth curve. Under the light condition, the constructed bacteria are difficult to grow in the test tube. The OD600 value maintained at about 0.045. And under the dark condition, the OD600 value increased from 0.046 at first to 0.205 after 2 hours, the difference was 0.143. After that, we chose to culture the bacteria under dark conditions for 5 hours and then change it to light conditions. The results showed that when the strain was cultured under light conditions for the first 1 hour, the lysis efficiency of the strain was high. The OD600 value decreased from 0.277 to 0.180, and the difference was 0.097. This indicated that the transformation of strains from dark to light conditions would cause lysis of bacteria.

(2) After the test tube experiment, we put the bacteria into the shaking flask to expand the culture. When cultured in shaking flask, dissolved oxygen increased. This is more benefit to the growth of bacteria. So, the maximum OD600 value also increased. After 30 hours of light culture, the OD600 value of bacteria decreased to 0.12. And under the dark condition, the OD600 value is about 0.997. The degradation rate reached 87.39%.

Reference

Geyi Wang, Xin Lu, Yisha Zhu, Wei Zhang, Jiahui Liu, Yankang Wu, Liyang Yu, Dongchang Sun, Feng Cheng (2018) A light-controlled cell lysis system in bacteria. Journal of Industrial Microbiology & Biotechnology https://doi.org/10.1007/s10295-018-2034-4

Hongping Wei, Hang Yang, Junping Yu (2015) A Kind of Lyase from Endolysis Escherichia coli and Its Application CN 104762285 Sequence and Features