8.17:

1. Preparation of LB medium

2. Production of gRNA-lacZ PCR system

3. Agarose gel electrophoresis

4. PCR products were recovered using a DNA extraction kit and the concentration was determined

5. Nanondrop concentration measurement

8.18:

1. Demethylation of gRNA-lacZ

2. Inactivation of DpnI

3. PCR product recovery

4. Nanodrop to measure concentration

5. Preparation of PCR system for Cas9-OP and ColE1

6. Transformation of Escherichia coli DH5a competent cells

8.19:

1. Select monoclonal identification and shake evenly for verification

2. Preparation of PCR system

3. Agarose gel electrophoresis

4. Monoclonal colonies were transferred for culturing

8.20:

1. Amplification of homologous arms through PCR

2. Plasmid extraction (gRNA-gntT)

3. Electrophoretic detection of extraction results

4. PCP product recovery

8.21:

1. Expanded culture and induction of p15A-Cas9

2. Homologous recombination ColE1-Cas9 plasmid

3. Agarose gel electrophoresis

4. PCR product recovery

5. Measure the concentration of product recovery results by nanodrop concentration measurement

8.22：

1. Extraction of crude protein by ultrasonic crushing

2. Protein purification

3. Configuration of LB liquid culture medium.

8.23：

1. Configure the protein gel and electrophoresis

2. Extract the plasmid

3. Measure the concentration of plasmid extraction

4. Run SDS-PAGE

8.24

1. Analysis of SDS-PAGE results

2. Bradford method for protein concentration (standard curve section)

3. Draw a standard curve

4. PCR products lambda-Red plastic recycling

9.16

1. PCR system for cas9-op and 15a fragments

2. PCR products were recovered using a DNA extraction kit and the concentration was determined

3. Homologous recombination 15A-Cas9-op plasmid

4. Nanondrop concentration measurement

5. Transformation of Escherichia coli DH5a competent cells

9.17

1. Select monoclonal identification and shake evenly for verification

2. Preparation of PCR system

3. Agarose gel electrophoresis

4. Monoclonal colonies were transferred for culturing

9.18:

1. Expanded culture and induction of p15A-Cas9-op

2. Extract the plasmid

3. PCR product recovery

4. Measure the concentration of plasmid extraction

5. Prepare Nissle 1917 competent cells

9.20：

1. Extraction of crude protein by ultrasonic crushing

2. Protein purification

3. Run SDS-PAGE

4. Bradford method for protein concentration

5. PCR amplifies the upstream homology arm and downstream homology arm of gnTt and lacZ, and overPCR connects the homology arms.

9.21:

1. Analysis of SDS-PAGE results

2. Nissle 1917 Electroporation with 15A-Cas9-op and 15A-Cas9, respectively

9.22:

1. Nissle 1917 Electroporation with sgRNA and homology arms, respectively

9.25/26:

1. Nissle 1917 Editing efficiency test