Experiment

●Plasmid Extraction

●Steps:

1)Take 1~5ml of bacterial liquid cultured overnight (12-16h), add it to a centrifuge tube, and centrifuge at 10000rpm for 1min. Discard the culture medium and invert it on absorbent paper to absorb the residual liquid.
2)Add 250ul of Buffer P1 to the centrifuge tube with the cell pellet (check whether RNase A has been added to Buffer P1 first), and mix with a pipette or vortex.
3)Add 250ul Buffer P2 to step 2, and gently turn the tubes upside down for 8-10 times to fully lyse the bacterial cells.
4)Add 350ul of Buffer P3 to step 3, and immediately turn the tubes upside down gently for 8-10 times to completely neutralize Buffer P2. Centrifuge at 12000rpm for 10min.
5)Place the FastPure DNA Mini Columns in the 2ml collection tube. Transfer the supernatant from step 4 to the adsorption column carefully with a pipette, taking care not to suck up the pellet, centrifuge at 12000rpm for 30-60s. Discard the waste liquid in the collection tube and put the adsorption column back into the collection tube.
6)Add 500ul Buffer PW1 to the adsorption column. 12000rpm, centrifuge for 30-60s. Discard the waste liquid and put the adsorption column back into the collection tube.
7)Add 600ul Buffer PW2 (check if it has been diluted with absolute ethanol) to the adsorption column. 12000rpm, centrifuge for 30-60s. Discard the waste liquid and put the adsorption column back into the collection tube.
8)Repeat step 7.
9)Put the adsorption column back into the collection tube. 12000rpm, centrifuge for 1min. Dry the adsorption column to completely remove the residual rinse solution.
10)Place the adsorption column in a new sterile 1.5ml centrifuge tube. Add 30-100ul Elution Buffer to the center of the membrane of the adsorption column. The DNA was eluted by centrifuging at 12000 rpm for 2 min at room temperature.
11)Discard the adsorption column and store the DNA product at -20°C to prevent DNA degradation.

Agarose Gel Electrophoresis

1)Prepare a 1% agarose gel with 1xTAE, and after the gel has solidified, place it into an electrophoresis tank filled with buffer.
2)Add 10×Loading Buffer to the sample at 1/10 of the loading volume, mix well and add to the gel pores.
3)Start the electrophoresis at a constant voltage, and analyze the DNA patterns with a gel imager.

●PCR

1)Design the primers for PCR amplification of the bpsA gene, and add the corresponding amount of water to the primer dry powder according to the instruction booklet.
2)Take 10µL of solution and add 90µL of water to prepare 10µmol/L primer solution for PCR.
3)After making the PCR system solution, put it into the PCR machine after shaking, and start the PCR machine to complete the amplification.

●Double Enzyme Digestion

1)Prepare multiple replicates of the digestion system solution in PCR tubes.
2)Seal with parafilm
3)After shaking tubes, enzymatic hydrolysis reaction takes place for 3 hours in a water bath at 37℃

●Linking Reaction

1)Prepare the solution in PCR tubes.
2)Seal with parafilm
3)After shaking tubes, put them in a constant low temperature water bath at 14℃ for linking reaction to take place.
4)Store at -20°C

●Culture and transformation of Escherichia coli

1) Cultivation of Escherichia coli

1. What is Escherichia coli?

Escherichia coli is a gram-negative, facultatively anaerobic enterobacterium

2. Preparation of Medium

E. coli can grow in common media, such as LB, SOB, Terrific media, 2xYT and other rich media. The higher the density of bacterial growth, the correspondingly more plasmid DNA will be obtained.

Generally, LB liquid medium is used to cultivate Escherichia coli. The following are the configuration steps of this medium:

a) Weighing: accurately weigh each component

Preparation: peptone 0.5g, yeast extract 0.25g, sodium chloride 0.5g, add water 50ml and 1g agar.

b) Dissolving: Use an alcohol lamp to heat and melt, dilute the volume to 50ml with distilled water or deionized water, and continuously stir with a magnetic stirrer throughout the process to prevent the agar paste bottom beaker from breaking.

c) Adjust pH value: add 1mol/L conc. sodium hydroxide solution to adjust pH to alkaline

d) Sterilization: Fill two 250ml Erlenmeyer flasks with 50ml LB liquid medium and 50ml LB solid medium respectively, and add cotton plugs. Wrap the petri dish with c raft paper, put it in a sterilizer, and sterilize 1kg under pressure for 15min.

e) Pour plate: After sterilization, operate near the flame of an alcohol lamp when the solid medium is cooled to about 60°C. Put the petri dish next to the flame, hold the triangular flask with the medium in the right hand, and pull out the cotton plug with the left hand; hold the triangular flask in the right hand, so that the mouth of the triangular flask quickly passes through the flame; open the petri dish with a left hand slightly larger than the mouth of the triangular flask Pour the medium into the petri dish with the right hand, and cover the dish immediately; after the plate is cooled and solidified, turn the plate upside down.

The purpose of placing it upside down is to prevent the water droplets formed during the cooling process of the medium from falling on the surface of the medium. When transferring sterilized medium to a petri dish, do not stick the medium on the walls of the dish. Otherwise, airborne bacteria will multiply on these adherent media and contaminate the media in the dish

3. Preparation of plates:

a) Weigh 10.0 g of tryptone, 5.0 g of yeast powder, and 10.0 g of NaCl, add 800 mL of secondary water to dissolve, stir evenly with a glass rod, adjust the pH to about 7.4 with 1 mol/L NaOH, set the volume to 1 L, and adjust pH 7.4 (if the pH of the solution is greater than 7.4, adjust with 1 mol/L HCl).

b) Distributed in conical flasks, the amount of each bottle should not be too much, about a finger below the bottom of the bottle. If solid medium is needed, add about 2% agar (2.5g agar to 150mL liquid medium) into the liquid medium after dispensing.

c) Cover the mouth of the conical flask with a plastic film with filter paper ventilation holes and hard paper in turn, and tie it with a rubber band. All Erlenmeyer flasks were operated as above. Use a marker to indicate the name of the medium and the date of preparation.

d) Sterilize in an autoclave at 121 oC for 15 minutes.

e) Adjust pH to detect the pH of the medium, if the pH is too acidic, add 1mol/L NaOH dropwise, stir while adding, and use pH test paper to test at any time until the desired pH range is reached. If it is too alkaline, adjust it with 1mol/L HCl. Adjustment of pH is usually placed before adding agar. It should be noted that the pH value should not be over-adjusted, so as to avoid callback and affect the concentration of each ion in the medium.

f) Filtration Liquid medium can be filtered with filter paper, and solid medium can be filtered with 4 layers of gauze while hot to facilitate the observation of culture. But for the medium for general use, this step can be omitted.

g) Packing According to the experimental requirements, the prepared culture medium can be packed into test tubes or triangular flasks. A funnel can be used when dispensing to avoid contamination caused by the medium sticking to the mouth of the tube or bottle.

h) The sterilized culture medium is taken out and dried in an electric blast dryer at 60oC, and taken out after the sealing paper of the conical flask is dry. The liquid medium can be stored or used directly. At this time, the medium with agar will not solidify. The medium can be poured into a petri dish on a sterile operating table that has been pre-sterilized by ultraviolet light for more than 30 minutes. About 10-15mL (90mm diameter), about 4mm thick in a petri dish. Stack the plates on the sterile operating table and place them for about 10 minutes. When the agar is basically solidified, the plates can be spread.

i) If the plate is not used directly, store the medium in an Erlenmeyer flask after sterilization. When the plate needs to be prepared, heat it in a microwave oven for about 3 minutes to melt the agar, and cool it at room temperature for 20 minutes until it is not hot to prepare the plate.

4. Inoculation of E. coli

Use the slab scribing method. The plate streaking method is a continuous streaking operation on the surface of the agar solid medium through the inoculation loop, and the aggregated strains are gradually diluted and dispersed on the surface of the medium. After several streaks, a single strain can be isolated.

Take the Escherichia coli BL21 cryopreserved in the laboratory, burn the mouth of the tube with an alcohol lamp, and open the centrifuge tube.

Inoculation method: Use a sterilized pipette tip dipped in the cryopreservation solution to draw horizontal strips on the edge of the plate, each three as a group, rotate the plate once, and finally make a zigzag in the middle. Because the experiment generally requires picking a single colony, the plate is adapted to consider the number of bacteria in the cryopreservation solution. If the amount of bacteria is too large, it should be appropriately diluted. General method one is more likely to obtain a single colony. Plate coating should be carried out near an alcohol lamp. If the plate coated with cryo solution should be turned upside down to prevent the generation of water vapor on the plate cover.

5. Culture and storage

The inoculated plate was inverted and placed in a constant temperature incubator at 37°C for cultivation, and colonies grew after about ten hours. Pick out a single colony with good growth status and obvious characteristics, inoculate it in freshly sterilized LB liquid medium at 37°C, and cultivate with constant temperature shaking at 220rpm/min for 9-12h. Colony characteristics: milky white, round, the colony edge is neat, the surface is smooth, and the surface and back are the same color.

2) Transformation of E. coli

The use of heat shock transformation

The experiment takes the following process:

1. Add the plasmid or DNA ligation product to be transformed i nto heat shock competent cells, mix well and place on ice. Plasmids were placed for 5 min, and the products were ligated for 30 min.

2. Place in a water bath at 42°C for 90 s, quickly place on ice, and place for 5 min.

3. Add 800 µL of antibiotic-free LB liquid medium, place at 37°C and incubate at 180 rpm for 45-60 min.

4. Apply an appropriate amount of bacterial solution to the LB plate containing the corresponding antibiotics, and invert overnight in a 37°C incubator.

5. Commonly used antibiotic screening concentrations: ampicillin: 100 mg/L, kanamycin: 50 mg/L

●Culture and transformation of Corynebacterium glutamicum

1)Cultivation of Corynebacterium glutamicum

1. What is Corynebacterium glutamicum

Corynebacterium glutamicum is a Gram-positive, a rod-shaped bacterium that is used industrially for large scale production of amino acids. While originally identified in a screen for organisms secreting L-glutamate, mutants of C.glutamicum have also been identified that produce various other amino acids.

2. Preparation

During the fermentation process of Corynebacterium glutamicum, sterile air should be continuously introduced, and by stirring the air to form fine air bubbles, which are quickly dissolved in the culture medium (dissolved oxygen); the temperature is 30 to 37 degrees Celsius, and the pH is In the case of 7 to 8, after 28 to 32 hours, a large amount of glutamic acid will be produced in the culture medium. In the production of glutamic acid, when the carbon-nitrogen ratio in the medium is 4:1, the glutamic acid produced by bacterial mass reproduction is less; when the carbon-nitrogen ratio is 3:1, the bacterial reproduction is inhibited, but glutamate The amount of acid synthesis is greatly increased. During the fermentation process, when the pH is acidic, Corynebacterium glutamicum will produce acetylglutamine; when the dissolved oxygen is insufficient, the metabolites produced will be lactic acid or succinic acid.

2) Transformation of Corynebacterium glutamicum

The use of electroporating method of cells. The experiment takes the following process:

1. Making electrocompetent cells

a) Grow cells in LBG medium at 30 °C and shaking at 200 rpm to an O.D.600 of 0.15 to 0.25.

b) Harvest by centrifugation.

c) Wash in one culture volume of 15% glycerol.

d) Resuspend cells in 0.002 culture volumes of 15% glycerol (number of cells: approx. 2.5 x 1010 cells/ml).

2. Electroporation of cells

a) Add 1-2 µl plasmid DNA (in water) to a minimum of 40 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette.

b) Wipe moisture from the cuvette and insert the cuvette into the device.

c) Electroporation:

                                                                                                     

Mode Prokaryotes	Voltage	Time constant
〇	2500V	5ms