1. strain

E.coli) K 12SF+ 02sf+

2. Culture medium

1) LB liquid medium: 10 mL/ bottle *4.

2) 2 times pounds of liquid medium: 3 ml/tube *2

3) LB solid medium: 250 mL/ bottle * 1.

4) Basic solid medium: 300 mL/ bottle * 1.

5) Nitrogen-free basal liquid medium: 5 mL/ bottle * 1.

6) 2N basic liquid medium: 5ml/ tube * 1.

Mixed amino acids and vitamins

Amino acids are divided into 7 groups, of which 6 groups have 6 kinds of amino acids in each group, and each amino acid is evenly ground and fully mixed (if the amino acid used is dl type, the dosage needs to be doubled).

Ⅰ

rely on

wrought

Methyl sulfide

cysteine

purine

cystine

Ⅱ

group

wrought

The abbreviation of Suzhou/Jiangsu Province/Soviet Union/a surname

kind

asparagus fern

pyrimidine

Ⅲ

The third/third in ten days' work

Methyl sulfide

The abbreviation of Suzhou/Jiangsu Province/Soviet Union/a surname

Hydroxyl reserve milk

sweet food

silk

Ⅳ

light

cysteine

kind

Hydroxyl reserve milk

Different brightness

Jacquard silk fabric

Ⅴ

Phenyl propyl

cystine

asparagus fern

sweet food

Different brightness

cheese

Ⅵ

colour

purine

pyrimidine

silk

Jacquard silk fabric

cheese

Ⅶ

Dried meat

Ⅷ

Mix vitamins (B 1, B2, B6, pantothenic acid, p-aminobenzoic acid, nicotinic acid and biotin are equally ground and fully mixed).

1. Preparation of bacterial liquid: Escherichia coli K 12 was inoculated in a triangular flask containing 10mlLB medium, and cultured at 37℃ overnight. 0.3ml flask inoculated with 10mlLB medium was cultured at 37℃ for 5 hours. Subpackage in two 5ml centrifugal tubes at 8000rpm for three times; Discard the supernatant, add 2ml of normal saline, and mix well to make 4ml of bacterial suspension.

2. Ultraviolet mutagenesis: pour 3ml of bacterial suspension into a small Petri dish (7cm), put the Petri dish under an ultraviolet lamp, sterilize it together with the cover for 65438±0min, and then open the cover and irradiate it for 3-4min. Add 3 ml of 2 lb culture medium, and then culture in a black box for more than 12 hours.

3. Inactivation of wild type with penicillin: take 3ml of bacterial liquid, centrifuge at 8000r/min for 3 minutes, and collect bacteria. Add 4ml of normal saline and centrifuge for three times to make 3ml of bacterial liquid. Take 0. 1 ml, add nitrogen-free basic medium (5 ml), and culture at 37℃ 1.2 hours or more. Then, 5ml of 2n basal medium and proper amount of penicillin sodium salt were added and cultured at 37℃.

4. Defect detection and culture: 0. 1 ml 12, 16 and 24-hour cultures were coated (LB, a basic medium) and cultured at 37℃ for 36-48 hours. Select the group whose colony number on the complete medium greatly exceeds that on the basic medium, use sterile toothpick to jump off 200 colonies growing on the complete medium, and point them on the basic medium plate and the complete medium plate respectively until the line is basically complete. 37oC culture.

5. Re-certification: select colonies that are basically absent on LB medium, mark them on basic medium for re-certification, and keep them on complete medium for later use. Those that are not long after 24 hours are defective.

6. Identification: The defect type to be detected was grafted in 10mlLB and cultured at 37℃ 12- 16 hours. The bacteria were collected by centrifugation at 8000 rpm for 3 minutes, and then washed by centrifugation with 4 ml of normal saline. Make 4 ml bacterial suspension. Pour 1ml into a basic plate, divide the bottom of the plate into 9 grids, put amino acids and vitamins in turn with an inoculation ring, 1 grid as a control, and culture at 37℃ for 2 hours without adding any nutrients.

It may be complicated. I'm sorry, I did my best.

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