Establishment Of A Genetic Transformation System For A Marine Chlorella Strain MEM 25

By creating and screening a（sub）tropical microalgal collection,we previously identified a Chlorella strain MEM25 with a robust growth in a wide range of salinities,temperatures,and light intensities.Evaluation of the economic viability and performance of different scale cultivation system designs（500 L and 5000 L closed photobioreactors and 60,000 L open race ponds,ORPs）at coastal zones under geographically specific conditions showed the stable and robust characteristics of MEM25 across different production system designs and various spatial and temporal scales.However,a lack of genetic transformation system of hinds an in-depth investigation of the marine chlorella,marine microalgal species in general.Therefore,employing this industrial microalga Chlorella strain MEM25 as a model,this study aims to establish a competent genetic engineering system for the marine Chlorella.First,the basic physiological characteristics of Chlorella sp.MEM25 were determined.Growth of MEM25 was monitored by detecting the turbidity(OD₇₅₀)at indicated intervals.With an initial inoculation concentration of 0.1(OD₇₅₀),the growth curve was relatively stable.The logarithmic growth period of MEM25 was from 72 h to168 h.A linear relationship between the optical density and cell numbers was plotted as y=13.148x+1.6312,R²=0.9911.We also optimized the cell number which could be hold on a single solid plate.The colonies distributed uniformly and appear as circular with a green coloration when 2×10⁵ algal cells were poured.Next,a dozen of different antibiotics were selected for the antibiotic sensitivity test.With a range from 0 to 1000μg/m L,MEME25 is not sensitive to kanamycin（Kan）,ampicillin（Amp）,cephalosporins drug（Cefo）,and hygromycin B（Hyg-B）.MEME25displayed a weak sensitivity to neomycin sulfate（NW）,spectinomycin（Spect）,streptomycin（Strep）,and gentamycin（Gent）while it was very sensitive to the zeocin,geneticin（G418）,chloramphenicol（chlorophyll）,and basta.The lethal doses of Chlorella to zeocin,basta,Chl,and G418 in liquid medium were 10μg/m L,80μg/m L,350μg/m L,and 550μg/m L,respectively.On the other hand,MEM25 was dead in 20μg/m L zeocin,200μg/m L basta,600μg/m L Chl,and 1000μg/m L G418,respectively in solid medium.The antibiotics Zeocin and the ble gene assembly were selected for further experiments.Because MEM25 was most sensitive to zeocin.Finally,the transforming cassette harboring e Ble gene in plasmid PMS188 was cloned and transferred into MEM25 by electroporation.The zeocin resistant strains expressing e Ble were obtained.PCR amplification and sequencing confirmed that the e Ble gene has been successfully introduced into MEM25.Multiple generations of subcultivation indicated that transformants were genetic stable.Then,expression cassette of the eble marker gene and the egfp reporter gene were simultaneously inserted into pBlue Script Ⅱ KS（-）vector by seamless DNA cloning technology.A vector nominated as PKS Ⅱ-eGFP-eBle was obtained and tried to be transformed into MEM25.In summary,in this study,we established a transformation system of marine chlorella strain MEM25.It holds a great promise for the development of synthetic biology using MEM25 as chasis.