Protocols

1. Media for incubating Escherichia coli

LB medium (Luria-Bertani Medium), TB medium (Terrific-Bertani medium), and NBS medium and the optimized fermentation medium was individually used for pre-culture of E. coli, protein expression, and shikimic acid synthesis.

(1) LB medium:

• Tryptone 10 g/L
• Yeast extract 5 g/L
• NaCl 10 g/L
• Distilled water up to 1 L g/L

(2) TB medium:

• Tryptone 24 g/L
• Yeast extract 12 g/L
• Glycerol 4 g/L
• K2HPO4 2.31 g/L
• KH2PO4·3H2O 16.42 g/L
• Distilled water up to 1 L

(3) NBS medium:

• KH2PO4 3.5 g/L
• K2HPO4 5 g/L
• (NH4)2HPO4 3.5 g/L
• CaCl2·2H2O 15 mg/L
• MgSO4·7H2O 15 mg/L
• Vitamins B1 0.5 mg/L
• (Carboxymethyl) trimethylammonium hydrochloride 1 mM
• Trace element mixture 1 mL/L
• Distilled water up to 1 L

  Trace element mixture (dissolved in 0.1 M HCl):

• FeCl3·6H2O 2.4 g/L
• CoCl2·6H2O 0.3 g/L
• CuCl2 0.15 g/L
• KMnO4 0.3 g/L
• H3BO3 0.075 g/L
• MnCl2·4H2O 0.5 g/L
• Distilled water up to 1 L

(4) The modified fermentation medium:

• Yeast tract 10 g/L
• K2HPO4·3H2O 7.5 g/L
• MgSO4 0.24 g/L
• Ammonium iron (III) citrate 0.3 g/L
• (NH4)2SO4 4 g/L
• Citric acid monohydrate 2.1 g/L
• L-Phenylalanine 0.5 g/L
• L-Tyrosine 0.5 g/L
• L-Tryptophan 0.25 g/L
• Biotin 10 mg/L
• Trace element mixture 1 mL/L
• Distilled water up to 1 L

Trace element mixture (dissolved in 0.1 M HCl):

• (NH4)6(Mo7O24)·4H2O 0.0037 g/L
• ZnSO4·7H2O 0.0029 g/L
• H3BO3 0.0247 g/L
• CuSO4·5H2O 0.0025 g/L
• MnCl2·4H2O 0.0158 g/L
• Distilled water up to 1 L

2. PCR experiment

(1)Materials for 50 μL reaction solution (Table 1-2)

(2)Steps

1. Prepare the required solution.
2. Set up the PCR reaction program;

Step 1: Denaturation of template DNA: heat the template DNA to around 98℃ for 5 min;

Step 2: Annealing (refolding) of template DNA and primers: the temperature drops to about 55℃ after heating and denaturing the template DNA into a single strand;

Step 3: Extension of primer: primer conjugates with DNA template by DNA polymerase to synthesize a new DNA strand complementary to the template, with dNTP and target sequence as the reaction raw material and the template, respectively;

Step 4: Repeating processes. It takes 2-4 minutes to complete each cycle, and 2-3 hours can amplify the target gene by several million times.

(3) Perform electrophoresis detection

Step 1: Weigh agarose (100 ml of 1×TAE buffer and 1g of agarose);

Step 2: Add 1×TAE;

Step 3: Mix evenly;

Step 4: Heat and melt in a microwave oven;

Step 5: Add fluorescent dye (ethidium bromide);

Step 6: Pour in the adhesive board;

Step 7: Wait for solidify;

Step 8: Put PCR reaction solution in the electrophoresis tank to start electrophoresis;

Step 9: Image gel. Compare with marker for viewing the migration of DNA fragments, and obtaining the required results.

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3. Recombinant plasmids construction

1.  Pick up the competent cells from the -80℃ refrigerator;
2.   Insert it into an ice box and let it sit for 5 minutes, and then transfer the DNA sample and mix well;
3.  Heat shock for 90 seconds at 42℃;
4.  Put it back in the ice box for 5 minutes;
5.  Add 750 μL LB medium;
6.  Place it in a shaker at 37℃ for 1 hour.

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4. Colony PCR validation and sequencing

(1) Prepare PCR (50 μL)

(2)Pick up the colony for validation

• 1) Take out a total of 10 centrifuge tubes and transfer them into 1.5 μL LB broth in the sterile workbench, respectively;
• 2) Use pipette gun to transfer the bacteria growing on the plate into a centrifuge tube, and then insert it into the prepared PCR solution;
• 3) Stir well (note that each scratch has a gun tip);
• 4) Mark carefully;
• 5) Performing PCR.
• 6) Gel electrophoresis verification.

(3) Sample sequencing

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5. Knocking out genes by CRISPR-Cas9

(1) Procedures：

The CRISPR-Cas9 method is mainly divided into the following steps:

• 1) Construct plasmid pTargetF targeting the desired gene by design sgRNA sequence;
• 2) Construct recombinant plasmid containing the upstream and downstream homologous arms of the target gene;
• 3) Plasmid pCas9 containing cutting function is electrocuted into E. coli MG1655 competent cells and cultured at 30℃, and then preparing the competent cells;
• 4) Plasmid pTargetF containing specific sgRNA sequences and homologous fragments of genes are transferred into the above strain, and the positive colony is obtained by colony PCR;
• 5) Add IPTG for eliminating plasmid pTargetF;
• 6) Cultivate at 42℃ for removing plasmid pCas9.

(2) Obtaining the recombinant plasmid

• 1) The bacteria are collected by centrifuging at 12,000 rpm for 2 minutes;
• 2) Add buffer P1 for remove intracellular RNA;
• 3) Add buffer P2 for lysing cells;
• 4) Add buffer P3 for neutralizing buffer P2;
• 5) Add buffer PW1, PW2 for removing inclusion;
• 6) Obtain the purified plasmid

(3) Purification and recovery of fragments

• 1) Enzyme digestion of the recombinant plasmids;
• 2) Cut off the gel containing the target DNA fragment, and weigh the gel weight (remove the empty tube weight) after DNA electrophoresis;
• 3) Add equal volume GDP buffer. Bath at 50~55 ℃ for 7-10 min, and adjust the time according to the size of gel to ensure that the gel block is completely dissolved.
• 4) Collect droplets on the tube wall through brief centrifugation. Add the fragments and carrier solution into the corresponding adsorption column (700 μL), centrifuge at 12000 rpm for 1 minute, and discard the waste liquid;
• 5) Discard the filtrate and place the adsorption column in the collection tube. Add 300 μL GDP buffer to the adsorption column, let stand for 1 minute, and centrifuge for 12000 for 1 minute;
• 6) Discard the waste liquid and place the adsorption column in the collection tube. Add 700 μL GW buffer and centrifuge at 12000 rpm for 1 minute;
• 7) Repeat step 5;
• Discard the filtrate and place the adsorption column in the recycling header. Centrifuge at 12000 rpm for 2 minutes;
• 8) Place the adsorption column in a 1.5 ml sterilized centrifuge tube, add 20 μL ddH2O to the center of the adsorption column, and let it sit for 2 minutes. Centrifuge at 12000 rpm for 1 minute.

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6. SDS-PAGE assay

(1) Preparation of SDS-PAGE electrophoresis gel

1) Select two glass sheets (one smooth and the other with a concave layer) and clean them;
2) Use green clip to clamp and check the sealing of the glass panel. Inject ddH2O into the glass plate until the liquid overflows, observe the liquid level, and if it is stable, it is successful;
3) If the sealing is not good, there are two measures: sealing the bottom or adding a blue needle at the top;
4) Making gel (adding different amounts of ingredients according to different concentrations).

• (i) 12% separation gel (10 mL)
  ddH2O 3.8 mL
  30% acrylamide 4 mL
  1.5 M Tris-HCI (pH 8.8) 2.5 mL
  10% APS (Ammonium persulfate) 0.1 mL
  10% SDS 0.1mL
  TEMED 0.004 mL
  
• (ii) 5% concentrated gel (2 mL)
  ddH2O 1.4 mL
  30% acrylamide 0.38 mL
  1 M Tris-HCI (pH 6.8) 0.25 mL
  10% APS (Ammonium persulfate) 0.02 mL
  10% SDS 0.02 mL
  TEMED 0.002 mL
  
• (iii) Specific pouring steps
  Step 1: Prepare separation adhesive and pour 8 mL into the glass plate;
  Step 2: Pour in isopropanol and flatten the concave surface of the separation adhesive solution;
  Step 3: Allow to stand for 40 minute and wait for solidification;
  Step 4: Pour out the upper layer of isopropanol;
  Step 5: Prepare concentrated adhesive, pour 2mL onto the glass plate, insert 15 holes, and use a 1.5mm comb;
  Step 6: Let it stand for 40 minutes until it solidifies (after solidification, two layers of adhesive can be seen with obvious layering lines).
  

(2) Sample preparation

1) Collect the engineered strains and the control strain cultivated in TB medium overnight with the same amount of OD600;
2) Resuspend strains with ultra-pure water and lyase by ultrasonic cell crusher for 10 minutes;
3) The whole cell, supernatant and precipitate were collected by centrifugation;
4) Add 5× protein loading buffer and heat for 10 minutes
5) Load sample.

(3) SDS-PAGE electrophoresis

• 1) Take out the gel and install the vertical electrophoresis tank
• 2) 5×Tris-Gly buffer is poured into the electrophoresis tank；
• 3) Remove the comb and place the sample (control group and experimental group) at 10 microliters into the sample hole;
• 4) Electrophoresis for 120 minutes (80 V for 30 minutes, and following 120 V for 90 minutes).

(4) Imaging

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7. Shake flask fermentation for shikimate biosynthesis

1) The seed liquid of the engineered strains cultivated at overnight was transferred into 50 mL fermentation medium at the proportion of 1% (v/v);
2) When OD600 was about 0.6-0.8, 50 μL IPTG and 500 μL arabinose was added to induce the expression of the desired genes, and the cultivation temperature was adjusted to 30℃;
3) Calcium carbonate is added during the fermentation process to control pH of the fermentation medium.

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8. Analytical methods

(1) Strain growth

The absorbance of the wavelength of 600 nm (OD600) was determined by spectrophotometer.

(2) Residue glucose concentration

• 1) Separating the supernatant from fermentation culture by centrifuging at 12000 rpm for 5 min;
• 2) Dilute it 100 times with ultra-pure water, and add 220 μL DNS solution at dark condition;
• 3) Heat 5 minutes after mixing up;
• 4) Add 800 μL ultra-pure water after cool before placing it on the special plate of the microplate reader;
• 5) Determine the absorbance of the wavelength of 540 nm, and convert the formula to obtain the glucose concentration.

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