NOTEBOOK

January：

Recruiting of new members
Theoretical knowledge learning and literature review

February：

Recruiting of new members
literature review
Project design

March：

Project design
Safety training

April：

Knowledge learning of synthetic biology
Writing of project plan

May：

Writing of project plan
Experimental operation and laboratory safety learning

June：

Enter the laboratory and start the experiment

July：

Synthetic biology experiment
Experimental data analysis
Education
Expert Interview Guide

August：

Discussion and solution of experimental problems
Building the Team Wiki
Education

September：

Data collation and analysis
Building the Team Wiki
Education

October：

Project arrangement and preparation
Drawing of experimental results

Stage 1 experiment
- Prepare LB for culturing competent cells
- Extract plasmid for target gene
- PCR extracted plasmid

1. LB preparation
- Tryptone 1 g
- Yeast extract 0.5 g
- NaCl 1 g
- Water 100 mL
Transfer to 2 conical flask
Add 1.5 g Agar in to one flask
Stored at 4 °C

2. Plasmid extraction
- UreB
- Nb-human
- Nb-6
- Nb-SCFV
- Centrifuge
- 1.5 ml centrifuge tube *4
- Buffer S
- SP1
- SP2
- SP3
- DW1
- Wash solution
- Elution buffer

3. PCR
- PrimeSTAR Max Premix 25 μL
- Primer 1 2.5 μL
- Primer 2 2.5 μL
- Tamplate 1 μL
- Water 19 μL

Stage 2 experiment
- Digestion of plasmid and target DNA
- Ligation of plasmid and target DNA
- Agarose gel electrophoresis
- Transfer to competent cells

1. Digestion
- 10x restriction buffer 5 μL
- HindIII 1 μL
- NheI 1 μL
- Plasmid DNA and the PCR result 1 μL
- Water 40 μL

2. Agarose gel electrophoresis
- TAE buffer 100 mL
- agarose 1.2 g
- Samples(plasmid,PCR production) 20 μL
- Electrophoresis tank
- Marker 20 μL
Run for 30 min at 120V

3. DNA gel extraction
- Target band(1.7kb and5.4kb)
- Buffer B2
- Wash solution 500 μL
- Elution buffer 20 μL
Stored at -20 °C

4. Ligation
- 10× buffer and T4 DNA ligase 10 μL
- Insert (1710 bp) and purified vector (5369 bp) 8 μL
The vector:Insert=1:3
Incubate for 2 h at the room temperature

5. Transfer to competent cells
- DH5α(cloning) 50 μL
- BL21(express) 50 μL
- ECN1917(Probiotics) 50 μL
- Ligation production 2μL each
- LB agar 900 μL
- Kanamycin 100 μL

Result:

- Gel electrophoresis results showed bands at 1710 bp and 5369 bp after comparison with the marker, but the colours were faint, that means some plasmids and PCR products were successfully digested.

- The fact that competent cells can grow successfully on medium containing kanamycin indicates that the constructed plasmid has been transferred into the cells, but further confirmation is needed to determine whether the plasmid can be expressed.

Stage 3 experiment
- Monoclonal colony identification and selection
- Colony PCR
- Agarose gel electrophoresis
- Expand BL21 for expression
- IPTG-induced expression

1. Colony PCR
- 2× Taq Premix 10 μL
- Primer 1 μL
- Primer 2 1 μL
- Colony
- Water 7 μL
Cycle of heat

2. Colony PCR verification
- Agarose 1.2 g
- Water 100 mL

3. Expansion of BL21
- LB 100 mL
- Kanamycin 100 μL
Shake for 1 h at 37 °C

4. Protein expression
- IPTG 100 μL
- BL21
- LB 100 mL
- Kanamycin 100 μL
Shake for 1 night

Result
- The results of gel electrophoresis were not ideal. All bands were at the bottom. We speculated that self-ligation may have occurred, or that the target gene was not inserted, or that some error occurred during the PCR process. However, after comparing our results with those of another group's repeated experiments, we speculated that the PCR failure may have been caused by enzyme denaturation.

We changed the experimental materials and conducted the experiment again. After optimizing the experimental scheme, we conducted the experiment again, and finally we got the correct PCR product.

Then, in order to obtain the protein we need, we added IPTG to the culture medium for protein.

Stage 4 experiment
- Ultrasonic disruption and protein extraction
- SDS-PAGE to verify protein

1. Protein extraction
- expanded BL21
- Centrifuge
- Ultrasonic crusher
- Lysozyme 50 μL
- BeyoGold His-tag 1000 mL
Repeat twice
- Lysis buffer 500 μL
- Elution buffer 500 μL
- Chromatography column

2. SDS-PAGE
- Agarose gel electrophoresis
- Lysis buffer 15 μL
- Marker(10-180KDa)
- Flow through
Wait 40 min under 120V
- Coomassie Brilliant Blue
Shake until protein stained

Result: The eluent is clearer than the flow-through liquid, indicating that impurity proteins have been filtered out.

Stage 5 experiment
- BCA protein concentration determination
- Western Blot

1. BCA
- BSA
- BCA regent A
- BCA regent B
Regent A:Regent B=50:1
- Extracted protein 20 μL each
Add lysis buffer until 20 μL
- Protein standard preparation solution 200 μL
- ELISA reader
Incubate at 37 °C for 30 min

2. Western Blot
- SDS-gel
- Splint
- Filter paper for transfer 2 sheets
- PDFV
- Methanol
- Sponge
- Transfer solution
- Electrospining tank
Add ice,powered on 150 mA for 80 min
- Blocking solution(5% BSA and BPST)
Add blocking solution in the PDFV for 30 min
- Mouse anti-his antibody
Wash extra antibody 3 times,10min per time
- Rabbit Anti-Mouse IgG H&L HRP secondary antibody
Incubating 1h at 4°C

Result

- We can estimate the relationship between protein concentration and absorbance based on the standard protein concentration. Then, we can calculate the protein concentration for each group of proteins using their absorbance values. The results are shown in the table above.

- The sizes of each protein:human：34.1kDa UreB: 59.5kDa nb-6 27.5kDa scfv： 44.8KDa
Each group has bands at the corresponding protein size positions, indicating that the proteins in each group are expressed. There are many bands in the eluent, indicating that there are many impurity proteins in the eluent. However, the protein bands in the first eluent are basically only close to the corresponding protein size, indicating that the proteins in the first eluent are relatively pure and that the target protein is present, but the specificity needs to be further verified.

Stage 6 experiment
- immunodiffusion test
- Determination of the inhibition rate of Hp Urease activity by recombinant nanobodies

1. Immunodiffusion test
- Agarose 1 g
- Water 50 mL
- UreB 1 mL
- Nb-human,Nb-6,Nb-SCFV 100 μL each
- Culture medium
- Oxford cup
-CK 100 μL
Incubate overnight at 37 °C

2. Determination of the inhibition rate of urease activity by recombinant nanobodies
- UreB 100 μg/mL
- Nb-6,Nb-human,Nb-SCFV
Incubate overnight at 4 °C
- Lysis buffer
- Urea solution 50 μL
- 96-well plate
- Phenol red 50 μL
Incubate at 25 °C for 6 h