Please design a plan to isolate an amylase producing strain from the soil, thank you

Transfer all microorganisms in the soil to the culture medium.

The medium uses starch as the whole C source, and the strains that cannot produce amylase cannot break down the starch, and there is no C source.

Cannot grow and reproduce.

Those that can produce amylase can multiply in large quantities and become the dominant strains in the culture medium.

As long as you prepare the starch screening plate, everything else is relatively simple.

1.Sample collection: You can design to focus on starch-rich plants, in which the bacterial starch decomposition plant isolation rate is relatively high, and you can also collect soil.

2.The sample was made into a suspension, and a certain amount was added to the enrichment medium (different enrichment medium can be prepared according to the situation, and nutrient broth, LB, etc.) can also be used), and the culture was left overnight.

3.The culture medium was diluted serially, a certain amount of starch plates were coated, and then cultured.

4., picking colonies with white transparent circles on the blue starch plate is amylase-produced.

Strains. 5.The amylase activity of each amylase-producing strain obtained was determined.

Pick high-yielding strains.

6.Classification and identification of high-yielding strains.

Amylase primary screening plates]: peptone, yeast extract. , soluble starch 1 g, trypan blue, agarose.

Adjust the pH to5 Or, add 100ml of DDH2O to medium autoclave for 20min, and pour the plate after warming.

First, the thallus is taken from the environment.

If the bacteria are cultured on a medium containing only starch and no glucose, the target strain can be grown.

If necessary, culture in the dark to eliminate the effect of photosynthetic bacteria.

Steps to produce amylase with genetically engineered strains.

Hello. Steps to produce amylase with genetically engineered strains:1

Select the amylase gene. The first step is to select the gene that encodes amylase, which is usually taken from the amylase-producing microorganisms, such as Bacillus, etc. 2.

Construct a recombinant plasmid. Genetic engineering technology was used to clone the amylase gene into the expression vector plasmid to construct the amylase recombinant plasmid. 3.

Transform host cells. The constructed recombinant plasmid was transformed into the host strain cells, and the host such as E. coli was transformed by electroporation or heat shock. 4.

Expression induction. Use an inducer such as IPTG to induce host cells to produce amylase, initiate a promoter in a recombinant plasmid, and express the amylase gene. 5.

Enzyme activity assay. DNS or other methods were used to detect the enzyme activity of amylase in the supernatant of the recombinant strain, and the successful expression of the enzyme was confirmed. 6.

Optimize expression. By changing the expression conditions and host strains, the expression amount of amylase was optimized, such as changing the temperature, vector copy number, etc. 7.

Purification and separation. The expression products were separated and purified by chromatography technology to obtain high-purity amylase products. 8.

Product preparation. The purified amylase is prepared into a commercial product by adding stabilizers and other stabilizers to adjust its properties, and is used in related industrial production. 9.

Save the strain. The recombinant strains were cryostored and identified to provide strains for the production of enzyme products in the future. Therefore, the main steps of producing amylase limb with genetically engineered strains include gene selection, expression vector construction, host transformation, expression induction, enzyme activity detection, enzyme expression optimization, purification and separation, and product preparation, which is a relatively complete production process.

The whole process needs to be carried out using molecular biology, genetic engineering, fermentation engineering and other technologies.