Developing a Small Molecule to Inhibit Phage
Infection of Bacterium Pseudomonas
Poster Presented by: SUSTech_Shenzhen 2020
Infection of Bacterium Pseudomonas
Zherui Li1, Jiaxi Zhang1, Shiqi Liang2, Linsheng Zhang2, You He3, Yunxuan Wang2, Linzhou Wang4, Ziyi Yin3, Yalikunjiang Aizezi2, Xvkun Yuan2, Yuao Zhu3
College of engineering, Southern University of Science and Technology, Shenzhen 518055, Guangdong, China
Department of Biology, Southern University of Science and Technology, Shenzhen 518055, Guangdong, China
School of medicine, Southern University of Science and Technology, Shenzhen 518055, Guangdong, China
College of science, Southern University of Science and Technology, Shenzhen 518055, Guangdong, China
Abstract: Sewage treatment is one of the most essential parts of water governing strategy. And among all microbes existing in the biofilm that used in wastewater treatment, Pseudomonas putida contributes vastly to waste processing due to its complex metabolism. The overgrowth of biofilms may block sewage treatment pipelines. An interesting idea arises that we can utilize phages to control the growth of biofilms. Meanwhile, stopping the excessive damage trend is also important. In our project, we aim to find a compound to protect Pseudomonas from phage infection through disrupting the expression of a critical transcription factor in phages.
To promote the usage of biofilm in sewage treatment
To shutoff the decline of Pseudomonas population caused by phages
To design a composite part for the screening of transcription factors inhibitors
2. The overgrowth of biofilms will block pipelines, while phages added artificially cannot be controlled and the biofilm used for sewage treatment will be completely destroyed. Therefore, we need to shutoff the damage process whenever we need.
3. We designed a composite part (BBa_K3423005), it was used for the screening of inhibitors against Aca1. One potential Aca1 inhibitor (Navitoclax) was found using this composite part, which proved the feasibility of this in vivo screening system, similar system can be derived form BBa_K3423005 for the screening of other transcription factors inhibitors.
In all, if the function of Aca proteins can be artificially inhibited via small molecule compounds, we will be able to increase the defense system of the bacterium against phages.
Engineering & Parts
Aca1 is constitutively expressed by T7 promoter, it will subsequently inhibit the anti-CRISPR (acr) promoter, so the gene expression downstream anti-CRISPR promoter will be inhibited until Aca1 inhibitor is added artificially. Without the inhibition from Aca1, the expression of genes downstream anti-CRISPR promoter will increase significantly. Therefore, protein expression can be regulated artificially. In this case, the activity of the acr promoter is estimated through measuring the intensity of green fluorescence.
Results & Conclusions
2. Then, 30 candidates we got form the virtual screening is tested using composite part BBa_K3423005, we applied our model to evaluate the efficiency of 10 different PDI inhibitor candidates and found that 1400-C4 has the smallest in vivo KI among those candidates. Therefore, we concluded that 1400-C4 could have the best inhibition efficiency and focused on 1400-C4 in the further experiment process. 3. 1400-C4 (Navitoclax) is tested for three times independently, R44A is the positive control, in which Aca1 is mutated to Aca1R44A, which lose its binding affinity to the acr promoter region. Data are represented as mean ± SEM from three independent experiments. 4. Navitoclax’s binding to Aca1 is verified using Differential Fluorescence Scanning Assay. 5. Based on the results from computer virtual molecular docking model of Navitoclax in complex with Aca1, we designed 60 inhibitors derived from Navitoclax, which is going to be tested latter. Conclusion: A promising hit of Aca1 inhibitor was found by SUSTech_Shenzhen from a very early step (target structure obtaining), a drug used to inhibit Pseudomonas phages will be found in the near future.
Application and Extension
Also, as our inhibitor can effectively protect Pseudomonas from phage infection, we propose that we can implement a strategy for Pseudomonas protection in the industry with massive cultivation of Pseudomonas, which is to add effective phage inhibitors during the cultivation.
Extension: With such a system, we can focus on another type of protein, the transcription factors that often act as the final executors of certain signaling pathways by either promoting or inhibiting the expression of certain sets of genes. Using a two-plasmid fluorescence-based reporter system, we are able to perform easy and convenient high-throughput chemical screening for inhibitors of transcription factors. And the corresponding small effector molecules could be utilized to exert precise control over the expression of certain sets of genes.
We focus on three main questions:
Is our idea of this project reasonable and does it have practical significance?
What can we do for improvement?
How can we develop a plan for an extension?
In the part of science communication, we mainly carried out three meaningful interaction activities. We designed an educational event for undergraduate students who are interested in biology, aiming to spread the concept of synthetic biology and make more students interested in synthetic biology and our project. Also, we produced and distributed science brochures on campus. Furthermore, we uploaded 2 small videos on the Bilibili website to introduce our project to the public.
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Liang Yang, Ph.D., Professor School of Medicine, Southern University of Science and Technology Mr. Shengchun Wu, Vice manager Shenzhen Techand Ecological and Environmental Co. Ltd. Hongwei Guo, Ph.D, Chair Professor Department of Biology, Southern University of Science and Technology QuanLiu, M.D, Ph.D, Associate Professor School of Medicine, Southern University of Science and Technology Kai Jiang, Ph.D, Research Assistant Professor Department of Biology, Southern University of Science and Technology Shaohong Zhang Undergraduate majoring in Biology Science, member of 2019 SUSTech_Shenzhen iGEM team Yi Zhang Undergraduate majoring in Biology Science, member of 2019 SUSTech_Shenzhen iGEM team Huazhang Shu Undergraduate majoring in Biology Science, member of Dr. Hongwei Guo’s Lab Shangzhen Yu Undergraduate majoring in Biology Science, member of Dr. Hongda Huang’s Lab Hongyu Bao, Ph.D Postdoc Fellow, Department of Biology Xiaoqian Xue, Ph.D Postdoc Fellow, Department of Chemistry SUSTech Presidential Foundation For generously providing funds for research.