Screening Of Mutagenic Mutations Of Aspergillus Oryzae Based On Microfluidic Chip Technology

As one of the important strains of traditional Chinese brewing,Aspergillus oryzae grows fast and has strong protein exocrine ability,so it is often used as an excellent host strain for foreign protein expression.As a screening marker for host fungus,auxotrophic marker genes are more efficient and safer.The microfluidic technology can quickly generate droplets of a certain size and size,which can be well applied to cell analysis.It greatly reduces the consumption of reagents and improves the screening speed.We use microfluidic system and traditional plate culture method to screen for pyrG-deficient strains.The main research contents are as follows:We built a microfluidic system platform and selected a flow-focused chip to generate droplets.The effect of different nonionic surfactants on droplet stability was studied.Observe the change of droplet size with time with a microscope,through the number of droplets,normalized average particle size,standard deviation of droplet size,normalized standard deviation of droplet size Determine the stability of the droplet.When the HLB value is about 5.5,the surfactant volume fraction is 5%,and the stability of the droplets is better in a short time.For a long time,we chose the oil phase composed of Abil EM90 to keep the droplets stable for 10 hours,so we chose Abil EM90 as the nonionic surfactant.Then,the relationship between the oil-water two-phase flow ratio and the droplet size and distribution is studied.When the ratio of Q oil/Q water is larger,the distance between the droplets in the flow channel becomes larger and larger,and the droplets are arranged without intervals until they present a linear distribution.As the Q oil/Q water increases,the droplet size becomes smaller and smaller.When Q oil/Q water is fixed,increasing the flow rate of the water phase will find that the droplets become larger and larger.Finally,we selected the program as Q water=0.054 mL/h,Q oil=0.22 mL/h,the droplets have a uniform spacing in the flow channel,the resulting droplet diameter is 98 ?m,and the stability is high.When optimizing the staining of nuclei with DAPI,it was found that the use of 5%formaldehyde as pretreatment and without pretreatment can make the staining effect good,and bright blue fluorescent nuclei can be observed under UV excitation light.It was observed in this experiment that Aspergillus oryzae spores contained 1-3 nucleus,and the spore diameter was distributed around 3-7 ?m.The average diameter of mononuclear spores was 3.1 ?m,the diameter of spores of two nuclei was 5.3 ?m,and the spores of three-nucleus The diameter is 7 ?m.Therefore,a 3 ?m diameter filter was chosen to enrich mononuclear spores.When the spore concentration was 5×105 cells/mL,we obtained a high percentage of single-cell monodisperse droplets with a probability of about 30%and a multi-cell droplet percentage of only about 6%.The spores started to germinate at the 6th hour of the droplet,and the mycelium had spread to the interface of the droplet after the 9th hour.Due to the difference in growth status between the spores,we chose to pick the mycelium after the 9th hour.Using Aspergillus oryzae H4 as the starting strain,it was mutagenized by plasma mutagenesis for 320 seconds.After microfluidic screening,a defective strain was initially named Aspergillus oryzae G1,which was identified by phenotype identification and gene sequence comparison.The results of trans complementarity verification showed that the defective strain could accept the complementary plasmid to restore its original phenotype.