Thermostable glucanase recombinant strain solid phase flat plate screening method

Abstract

The invention discloses a solid-phase plate screening method of heat-resistant dextranase recombinant strain, which comprises the steps as follows: a nylon membrane is sterilized at the temperature of 121 DEG C for 15 minutes; E.coli JM109BL21(DE3)pET- 20b-Cel12B is cultured in 5ml of LB liquid culture medium for 6 hours; the LB solid culture medium is coated with 4mul of IPTG with the concentration being 5mug/mL and keeps upright at the temperature of 37 DEG C for 2-3 hours; an LB solid plate is coated with strain solution, and cultured at the temperature of 37 DEG C for 15 hours; the plate is kept at the temperature of 4 DEG C for 1-2 hours; the nylon membrane is placed on the plate to photocopy colony; the colony surface affected by the nylon membrane is upwards placed on the LB solid plate and inversely placed and cultured at the temperature of 37 DEG C for 4-5 hours; the nylon membrane is soaked in the mixture of cell wall cracking solution, cell membrane cracking solution and equilibration buffer solution for 30min; the colony surface of the nylon membrane is downwards laid on a screening plate, and kept at the temperature of 70 DEG C for 3 hours; the nylon membrane is uncovered and the screening plate is developed by 0.1% of Congo red for 15min, and decolored by 1mol/L of NaCl for 15min to select transparent circle strains. The method has the advantages of being simple, saving time and avoiding trouble, high screening efficiency and good screening effect.

Description

technical field [0001] The invention relates to the fields of microbiology, genetic engineering, biotechnology and the like, in particular to a solid-phase plate screening method for extremely thermostable glucanase recombinant bacteria. Background technique [0002] The world generates 150-200 billion tons of plant-based organic matter every year, about half of which are cellulose substances. How to degrade cellulose substances into easy-to-use small molecular organic substances such as sugar, ethanol, and methane is one of the major international issues at present. . At present, chemical methods such as acid-base treatment and physical methods such as steam explosion and steam heating are mainly used for the degradation and utilization of cellulose. The biological method has attracted much attention due to its low requirements for equipment, easy recycling of decomposed products, and less environmental pollution. Because heat-resistant bacteria can grow in a high-tempera...

AI Technical Summary

Problems solved by technology

However, when using a 96-well plate to screen high-temperature-resistant enzymes, the colonies must be individually picked into the 96-well plate for cultivation, and then the culture solution is subjected to heat treatment and enzyme activity determination, which is time-consuming and laborious, and because the colored substrate used is very expensive. lead to higher testing costs

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