High Throughput Screening Of Heat Shock Resistant Strains Based On E.coli Expression Library And C.elegans Model

Caenorhabditis elegans is a tiny invertebrate and the first multicellular organism whose genome to be sequenced.Of note,60-80% genes of C.elegans are homologous to human genes and more than 40% genes are human disease-related genes.Heat shock response is known to be involved in many diseases,e.g.its protective role in neurodegenerative diseases.Here a high-throughput screening method using C.elegans as a model was explored to screen for E.coli strains expressing anti-heat shock proteins from sea cucumber cDNA library in 96-and 384-well microplates.1.Optimization of high-throughput screening platform and screening conditions.First,we explored the screening conditions such as screening volume,bacterial concentration and fluorescence detection time.Then we tested food preference of the nematodes in terms of different E.coli strains.We also tested image capture and analysis methods.The screening process involved a laboratory automation workstation,a COPAS biosorter and a high content screening system.The laboratory automation workstation was used for accurate addition of liquid in large scale with high reliability.The COPAS Biocort was used to to accurately add and sort C.elegans to the microplates.The high content screening system used to detect and analyze the fluorescence intensity of C.elegans.This combination was effective to reduce labor and human errors and thus increase experimental reliability and improves screening efficiency.2.High-throughput screening method for heat shock resistant strains.This work was performed using a sea cucumber cDNA library previously constructed in the lab.The library vector plasmid was introduced into E.coli and induced for expression.The E.coli that expressed the library protein was fed to C.elegans.After incubation for a specific period of time,SYTOX green fluorescent dye was added and the nematodes are heated shocked.The high content screening system was then used to image the nematodes through bright field and FITC channel and analyze the image data obtained.3.High-throughput screening methods for anti-aging and antioxidant strains.Lipofuscin,also known as senile pigment,increases with age,and is considered a hallmark of aging.Therefore,we attempted to screen E.coli strains expressing anti-aging protein by high-throughput detection of lipofuscin content in C.elegans.Reduction of ROS level can reduce the damage caused by oxidative stress and thus may delay aging,therefore we also attempted to screen strains expressing antioxidant proteins by detecting ROS level in C.elegans using the high-throughput system.In conclusion,a high-throughput screening method was constructed for heat shock resistant E.coli strains,which had the characteristics of high throughput and automation.