Strain improvement by UV

Experiment 15 Strain improvement by UV mutagenesis Date: Replica Plate technique was developed by Joshua and Esther Lederberg in 1952 for providing direct evidence for the existence of pre-existing mutations. This technique isolates both nutritional mutants and antibiotic-resistant mutants. Their actual experiment concerned with replicating master plates of sensitive cells to two or more plates containing either streptomycin or T1 phage. Replica plating allows the observation of microbes under a series of growth conditions. The bacteria are grown in an environment that is not selective for a given mutation. This technique is used to transfer the members of each colony to a selective environment. A simple velveteen covered colony transfer device is used to transfer the colonies in nutrient agar medium supplemented with or without a particular antibiotic or nutrient. The fibers of velvet act as fine inoculating needles, picking up the bacterial cells from the surface of this master plate. The velvet with its attached microbes is then touched to the surface of a sterile agar plate, inoculating it. In this manner, microbes can be repeatedly stamped onto media of differing composition. By comparing the presence of colonies following incubation, we can indirectly determine the mutant colonies by their absence in the selective environment. A colony that develops on a complete medium fail to develop on a minimal medium that lacks a specific growth factor (more often amino acid), the occurrence of a nutritional mutant is indicated. The microbes that do not grow on the minimal medium represent auxotrophic strains. Materials required:  24 h old nutrient broth culture of Escherichia coli.  Minimal salt agar with glucose.  Three 10ml Nutrient agar deeps. Procedure Part I: Mutation of E. coli with UV radiation AGM 312 Microbial Biotechnology (1+1) Page 63   Inoculate LB broth tube with E. coli and incubate it for 16 h at 37ᵒC Dilute the overnight culture 100 fold into fresh LB broth and grow to a cell density of 2 x 108cells per ml  Wash the bacteria by filtration or centrifugation and re฀suspend them in PBS to have a cell density of 2 x 108 cells per ml  Plate 0.1 ml aliquot of 10฀5 dilution in duplicate on solid LB agar medium  Incubate the plates at 37 °C for 24 to 48 h  Observe the plates for the development of E. coli colonies  Count the no. of viable cells per ml prior to irradiation  Irradiate the culture of E. coli with UV to get UV induced mutant of the bacterium  Pipette 5 – 6 ml aliquot of bacteria suspended in PBS as prepared in step 3 above, at 2 x 108 cells per ml to sterile Petri dishes.  Remove the top of Petri dishes and place them on the shaker underneath the light source at a distance of 30 cm, and expose the plates for 15 to 20 min  Transfer 1 ml of UV treated culture in 10 ml nutrient broth and incubate overnight in stationary phase at 37 °C  Pipette 0.1 ml of the undiluted as well as 101 or 102 fold dilutions of cultures on to nutrient agar plates i.e. complete medium  Spread the organism over the plates with a sterile bent glass rod  Incubate this plates in an inverted position at 37 °C for 24 h Part II: Screening auxotroph mutants by Replica Plate technique  Prepare agar medium with minimal medium with and without lysine  By holding the sterile colony carrier handle in the right hand, carefully lower the velvet surface and press on the master plate surface having colonies of E. coli both wild type and mutant survivor to pick up bacteria from each colony  Inoculate the minimal agar plate (i.e. complete medium minus lysine) by lightly pressing the velvet onto the medium to get a replica plate.  Now without returning the carrier to the master plate, inoculate the complete medium plate in the same manner  Incubate both the inoculated plates upside down at 37 °C for 48 ฀ 96 h Observation Examine both the minimal agar and complete medium plates for the appearance of E.coli colonies and identify those which grew on complete medium but not on the minimal agar plates. Determine the location of lysine auxotroph on the complete medium replica plates. Result and inference: AGM 312 Microbial Biotechnology (1+1) Page 64 Reference: S.Javed, M.Asgher, M.A. Sheikh and H. Nawaz. 2010. Strain Improvement Through UV and Chemical Mutagenesis for Enhanced Citric Acid Production in Molasses-Based Solid State Fermentation, Food Biotechnology, 24:2, 165-179, DOI: 10.1080/08905436.2010.482014 AGM 312 Microbial Biotechnology (1+1) Page 65