iGEM 2024 — Team zhejiang-united

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NOTEBOOK
![](https://static.igem.wiki/teams/5067/s5/99-1.png)

[Notebook](#nav1)

[Notes for 2024/6/9~6/16](#nav2)

[Notes for 2024/6/16~6/23](#nav3)

[Notes for 2024/6/23~6/30](#nav4)

[Notes for 2024/6/30~7/7](#nav5)

[Notes for 2024/7/7~7/14](#nav6)

[Notes for 2024/7/14~7/21](#nav7)

[Notes for 2024/7/21~7/28](#nav8)

[Notes for 2024/7/28~8/4](#nav9)

[Notes for 2024/8/4~8/11](#nav10)

[Notes for 2024/8/11~8/20](#nav11)

Notebook

A: anti-TNFa nanobody antibody—A (TNFa binding fragment of Ozoralizumab)

B: anti-TNFa nanobody antibody—B (TNFa binding fragment of V565)

Notes for 2024/6/9~6/16

**Overview**

This file contains all record of experiment from 2024/6/9 to 2024/8/30

**Experiment goals**

plasmid construction and molecular cloning of p15A-GEx-A/B

Participants

Kaitlyn, Hellen, Friedman, Steven, Eric, Amie, John, Sylve, Grayson, Kevin, DJL

Summary

* Gene synthesis of the nanoAb-A/B sequence by GeneScript.
* Preparation of culture medium of the bacterial, including liquid and solid form of LB and SB medium.
* PCR amplification of target gene A，B and linearization of vector backbone

Experiment process

**Gene synthesis:**

We download and verify the sequence of gene A and B then submitted to the gene synthesis company GeneScript.

**system:**

PCR amplification of A, B and vector backbone

-1ul of DNA template

-1ul of primers for P1 and P2 respectively

-10ul of Polymerase

-7ul of double distilled water

**PCR steps: 40× cycles**

1. Adjust the numbers with a pipette first, and then mix the materials

2. Inactivate at a high temperature of 98 °C Celsius, causing DNA to change from double stranded to single stranded

3. Deactivate 56 °C primer binding to DNA

4. 72 °C extended DNA pairing for 1 minute

5. Repeat about 40 cycles to obtain a large amount of DNA

**Make LB medium**

-add tryptone(1%), yeast extract(0.5%), NaCl(1%) into double distilled water and make it dissolved in it.

-sterilize the mixture at the temperature of 121 Celsius degree for 20 minutes.

- Add chloromycetin into the sterile medium and poured the solid LB medium into plates and allowed it to solidify

Results

Notes for 2024/6/16~6/23

Participants

Kaitlyn, Hellen, Friedman, Steven, Eric, Amie, John, Sylve, Grayson, Kevin, DJL

Summary

* Gel Electrophoresis and extraction to purify the target PCR fragment
* Homologous Recombination

Experiment Process

**Electrophoresis**

Prepare the TAE buffer:

-Add TAE buffer powder (sufficient for 1L).

-Add 1L of water and shake until fully dissolved.

-Weigh the agarose (1%) and mixed with TAE buffer then boiled in microwave.

-Add about 1:10000 amount of Gel Red to the melted agarose in the flask and continue to mix gently by swirling until the Gel Red is completely mixed with the melted agarose.

-Pour into a mold and allow it to solidify.

Load the Gel:

Once solidified, remove the gel from the mold and place it in the electrophoresis apparatus.

-Add marker to the first well.

Mix 4 μL of 6× loading buffer with 20 μL of sample, and load into alternating wells.

-Run the gel for 30 minutes.

Remove the gel and place it under UV light to document the results.

**extraction and purify the PCR product**

1. cut out the needed parts of the gel that contain DNA

2. add buffer B2 (3-6 times of the amount of the cut gel)

3. place the substances at 50℃ for 5-10 minutes

4. move the solution into a collection tube with a spin column

5. centrifuge 9000g 30sec

6. remove the flowed liquid, add 500ul washing agent and centrifuge 9000g 30sec

7. repeat step 6

8. centrifuge the spin column 9000g 1min

9. transfer the spin column into a clean 1.5 mL tube and add 15~40 uL Elution Buffer. place in room temperature for 1 minute then centrifuge for one minute.

10. Discard the spin column and preserve the flow-through DNA solution.

**Homologous Recombination and transformation of DH5a competent cell**

Mix 0.5 μL each of A/B+ backbone of vector.

Add 5 μL enzyme mix.

Incubate at 50°C for 5 minutes.

Transfer the reaction mixture to ice.

Transformation:

1. Prepare Cells:

- Use 100 μL of competent DH5α cells.

2. Mix and Incubate:

- Gently mix 10 μL of the reaction mixture with the competent cells.
- Incubate on ice for 30 minutes.
- Heat shock at 42°C for 45 seconds.
- Transfer back to ice for 2-3 minutes.- Add 900ul LB medium without antibiotics and incubate with shaking to allow recovery for 60 minutes

  3. Smear the transformed bacterial cell to culture plate

  - When the bacterial recovery is complete, centrifuge the tubes for 5 minutes at low speed.
  - Discard but leave 100 uL of supernatant left. Resuspend the cell pellet. Make sure the entire pellet is resuspended before plating.
  - Aliquot the entire 100 uL to an LB agar plate.
  - Incubate the plate at 37 degree for 24 hours.

Results

![](https://static.igem.wiki/teams/5067/note1/1.png)

First eletrophoresis: DNA marker is degraded and the result is faild

![](https://static.igem.wiki/teams/5067/note1/2-1.png)

Second electrophoresis: choose a new DNA marker and redo the PCR reaction

![](https://static.igem.wiki/teams/5067/note1/2-2.png)

Notes for 2024/6/23~6/30

Participants

Kaitlyn, Hellen, Friedman, Steven, Eric, Amie, John, Sylve, Grayson, Kevin, DJL

Summary

- Bacterial colony PCR to verify the successful engineered DH5a
- Colony PCR Gel electrophoresis
- Culture of the successful engineered DH5a overnight

Experiment Process

**Bacterial colony PCR and gel electrophoresis**

Step 1: We took out the Escherichia coil we cultured and put them into the PCR machine to verify not being mistaken in our experiment.

The culture disk on the left side is the bacterium that added DH5A with A-antibody, while the one on the right of this picture is added with B-antibody, CHR and DH5A.

Step 2: Use PCR technique and gel electrophoresis to verify the bacterium we cultured.

Take eight tubes of monoclonal bacterial strain by using the pipette tips. After that, we put 10 ul of sterile water and used the system of PCR including 2 ul of bacterial solution, 1 ul of primer number 1, 1 ul of primer number 2, 10 ul of mixture and 6 ul of double distilled water.

**load the product into gel**

Once solidified, remove the gel from the mould and place it in the electrophoresis apparatus.

Add marker to the first well.

Mix 4 μL of 6× loading buffer with 20 μL of the PCR sample, and load into alternating wells.

Run the gel for 30 minutes.

Remove the gel and place it under UV light to document the results.

The expected bands will be visible if successful, indicating successful plasmid construction.

**Culture of the successful engineered DH5a overnight**

After verifying the bacterium we cultured, we kept culturing the bacterium by adding 3 ml LB culture medium and CHR (1:1000) for each tube of bacterial solution.

Inoculate each tube with a small amount of the confirmed bacterial suspension.

Incubate overnight with shaking at 37℃ and 220rpm

Results

![](https://static.igem.wiki/teams/5067/note1/4-1.png)
![](https://static.igem.wiki/teams/5067/note1/4-2.png)
![](https://static.igem.wiki/teams/5067/note1/4-3.png)

A1, A2, A3, A4

![](https://static.igem.wiki/teams/5067/note1/4-4.png)

B1, B2, B3, B4

Notes for 2024/6/30~7/7

Participants

Kaitlyn, Hellen, Friedman, Steven, Eric, Amie, John, Sylve, Grayson, Kevin, DJL

Summary

- Plasmid extraction and sanger sequencing (third party)
- Analysis of the sequencing result to the designed plasmid sequence

Experiment Process

**extraction**

Preparation:

Add 500 μL of Buffer S to a spin column.

Centrifuge at 12000g for 1 minute.

Discard the flow-through from the collection tube.

Bacterial Cell Harvesting:

Add 2 mL of the overnight bacterial culture to an Eppendorf tube.

Centrifuge at 8000g for 1 minute.

Discard the supernatant.

Cell Lysis and Neutralization:

Add 250 μL of Buffer SP1 to the cell pellet and mix thoroughly.

Add 250 μL of Buffer SP2 and invert the tube 4 times to mix.

Add 350 μL of Buffer SP3 and centrifuge at 12000g for 5 minutes.

Plasmid Binding:

Transfer the supernatant to the prepared spin column.

Centrifuge at 8000g for 30 seconds.

Discard the flow-through and reapply the liquid to the spin column.

Centrifuge again at 8000g for 30 seconds.

Wash Steps:

Add 500 μL of DW1 to the spin column.

Centrifuge at 9000g for 30 seconds.

Add 500 μL of Wash Solution.

Centrifuge at 9000g for 30 seconds.

Repeat the wash step with 500 μL of Wash Solution and centrifuge at 9000g for 30 seconds.

Drying:

Centrifuge the empty spin column at 9000g for 1 minute to remove any residual wash solution.

Elution:

Transfer the spin column to a new 1.5 mL Eppendorf tube.

Add 50 μL of Elution Buffer to the center of the spin column membrane.

Let it stand for 1 minute.

Centrifuge at 9000g for 1 minute.

Reapply the eluate to the spin column and centrifuge at 9000g for another minute.

Remove the spin column and retain the Eppendorf tube containing the purified plasmid DNA.

**Analysis of the sequencing result to the designed plasmid sequence**- After sanger sequencing, we obtained two files for each plasmid A or B
- and we upload the .ab format file to the Snapgene software to blast with the origin sequence of p15A-GEx-A/B respectively for A and B
- We analysis and check the base match and find there is no mismatch for our constructed plasmid.

Results

![](https://static.igem.wiki/teams/5067/note1/5-1.png)
![](https://static.igem.wiki/teams/5067/note1/5-2.png)

Culture of successful engineered bacteria overnight

Sequence result of Nanobody antibody A

![](https://static.igem.wiki/teams/5067/note1/5-3.png)

Sequence of Nanobody antibody B

![](https://static.igem.wiki/teams/5067/note1/5-4.png)

Notes for 2024/7/7~7/14

Overview

This file contains all record of experiment from 2024/7/7 to 2024/7/28

Experiment goals

Engineered EcN1917 verification and culture for the collection of supernatant of nanoAb

Participants

Kaitlyn, Hellen, Friedman, Steven, Eric, Amie, John, Sylve, Grayson, Kevin, DJL

Summary

- Transformation of EcN-1917 for the plasmid p15-GEx-A and p15A-GEx-B
- EcN 1917 colony PCR verification

Experiment process

**Transformation of EcN-1917 for the plasmid p15-GEx-A and p15A-GEx-B**

Prepare Cells:

Use 100 μL of competent ECN cells.

1. Add 100 uL of competent cell (EcN1917) to the 1 uL constructed plasmid A or B seprately.
2. Place on the ice for 30 minutes.
3. After the ice incubation, heat shock the tubes at 42 degree for 90 seconds.
4. Return to the ice for 2-3 minutes.
5. Add 900 uL of LB broth to each tube.
6. Vortex each tube and incubate them at 37°C for 60 minutes while shaking.
7. When the bacterial recovery is complete, centrifuge the tubes for 5 minutes at full speed.
8. Discard but leave 100 uL of supernatant left. Resuspend the cell pellet. Make sure the entire pellet is resuspended before plating.
9. Aliquot the entire 100 uL to an LB agar plate.
10. Incubate the plate at 37 degree for 24 hours.

**EcN 1917 colony PCR verification**

1. Add 10 uL of LB broth in each tube and gently mix.
2. Obtain the plates and gently remove 6 bacteria colonies from the surface of the plate to sterile tubes.
3. Obtain a 0.2 mL tube strips and place on the bench.
4. Add 2 uL of each mixed solution, 1 uL of P1, 1 uL of P2, 6 uL of ddH2O, and 10 uL of PCR reaction mix in each 0.2 mL tube strips. The total amount of mixture in each tube of the 0.2 mL tube strips should be 20 uL.
5. Do a PCR of the tube in the PCR apparatus
6. After PCR, to check the plasmid is right or wrong.

Results

![](https://static.igem.wiki/teams/5067/note1/6-1.png)
![](https://static.igem.wiki/teams/5067/note1/6-2.png)

Notes for 2024/7/14~7/21

Participants

Kaitlyn, Hellen, Friedman, Steven, Eric, Amie, John, Sylve, Grayson, Kevin, DJL

Summary

- Make SB medium
- Culture of EcN 1917- A/B at LB/SB and 16/37 Celsius
- Filtration of the culture medium supernatant by 0.22um filter
- Reservation of the nanobody antibody A/B supernatant at -80℃

Experiment process

Make SB medium

Weigh and solve the various regent by ddH2O

Trypton 3.2%

Yeast extract 2%

NaCl 0.5%

**Culture of EcN1917- A/B at LB/SB at 16/37 Celsius and collect the supernatant of medium**   1. The    preceding night, the bacteria were inoculated into LB liquid    culture medium and incubated overnight. Chloramphenicol was    subsequently added to achieve a final concentration of 25    micrograms per milliliter, specifically for Nissle1917 expressing    antibodies A and B. 2. On    the following evening, measure the optical density (OD) and    select the strain with the highest OD value. Inoculate this    strain at a concentration of 1% of its volume into SB medium    containing chloramphenicol and adjust the volumes of the    remaining strains to match this maximum OD value. Based on the    added volume, proportionally increase the inoculation volume in    LB medium while incubating at 16°C and shaking at 220 rpm for 12    hours; prior to incubation at 37°C, measure the OD to ensure    that all inoculated liquid starts from an equivalent initial    volume. 3. Centrifuge    at 3000 g for 5 minutes at 4°C, collect the supernatant, filter    it through a 0.22-micron filter, and store the resulting solution    at -80°C. |

Results

![](https://static.igem.wiki/teams/5067/note1/7-1.png)

37 Celsius

![](https://static.igem.wiki/teams/5067/note1/7-2.png)

16 Celsius

Notes for 2024/7/21~7/28

Participants

Kaitlyn, Hellen, Friedman, Steven, Eric, Amie, John, Sylve, Grayson, Kevin, DJL

Summary

- WB to detect the expression of nanobody antibody A/B with His tag

Experiment process

**Materials Required:**   * **Protein   lysates** (containing   your samples) * **SDS-PAGE   gel system** * **Transfer   apparatus** (e.g.,   semi-dry transfer cell or tank transfer system) * **Nitrocellulose   membrane** or **PVDF   membrane** * **Blocking   buffer** (such   as 5% non-fat milk in TBS-T) * **Primary   antibody** (anti-His   tag antibody) * **Secondary   antibody** conjugated   with an enzyme (such as HRP-conjugated anti-mouse IgG if the   primary is mouse-derived) * **ECL   substrate** for   chemiluminescence detection * **Imaging   system** (e.g.,   ChemiDoc MP Imaging System)   **Procedure:**  **Step 1: Sample Preparation**   1. **Prepare    protein lysates**:    Ensure that your lysates contain a reducing agent like DTT or    β-mercaptoethanol. 2. **Boil    samples**: Boil the    lysates at 95°C for 5 minutes to denature the proteins and allow    them to bind to the SDS.   **Step 2: Electrophoresis (SDS-PAGE)**   1. **Load    samples**: Load    equal amounts of protein lysates into the wells of the pre-cast    polyacrylamide gel. 2. **Run    the gel**: Apply a    constant voltage (e.g., 80V for stacking gel, then increase to    120V for resolving gel) until the tracking dye reaches the bottom    of the gel.   **Step 3: Transfer (Blotting)**   1. **Assemble    transfer sandwich**:    Place the nitrocellulose or PVDF membrane on top of the gel,    ensuring there are no air bubbles. 2. **Perform    transfer**: Use    either a semi-dry transfer apparatus or a wet transfer tank.    Apply a constant current (e.g., 300mA for 90 minutes) to transfer    the proteins from the gel to the membrane.   **Step 4: Blocking**   1. **Block    non-specific binding sites**:    Incubate the membrane in blocking buffer for 1 hour at room    temperature or overnight at 4°C to prevent non-specific antibody    binding.   **Step 5: Primary Antibody Incubation**   1. **Prepare    primary antibody**:    Dilute the anti-His tag antibody in blocking buffer according to    the manufacturer's instructions. 2. **Incubate**:    Incubate the membrane with the diluted primary antibody for 1-2    hours at room temperature or overnight at 4°C.   **Step 6: Wash**   1. **Wash    the membrane**: Wash    the membrane several times with TBS-T (Tris-buffered saline with    Tween-20) to remove unbound primary antibody.   **Step 7: Secondary Antibody Incubation**   1. **Prepare    secondary antibody**:    Dilute the HRP-conjugated secondary antibody in blocking buffer. 2. **Incubate**:    Incubate the membrane with the diluted secondary antibody for 1    hour at room temperature.   **Step 8: Final Wash**   1. **Wash    the membrane**: Wash    the membrane several times with TBS-T followed by a final wash    with TBS alone.   **Step 9: Detection**   1. **Apply    ECL substrate**:    Apply the chemiluminescent substrate to the membrane. 2. **Develop    the signal**: Place    the membrane in a cassette and expose it to film or use a digital    imaging system to visualize the bands.   **Step 10: Analysis**   1. **Evaluate    results**: Analyze    the bands using software such as Image Lab to confirm the    presence and size of the His-tagged protein. |

Results

![](https://static.igem.wiki/teams/5067/note1/8.png)

Notes for 2024/7/28~8/4

Overview

This file contains all record of experiment from 2024/7/28 to 2024/8/4

Experiment goals

ELISA1: expression test of nanobody antibody A/B

Summary

Kaitlyn, Hellen, Friedman, Steven, Eric, Amie, John, Sylve, Grayson, Kevin, DJL

Experiment process

ELISA assay：TNFa coated plate   1. A    final concentration of 100 ng/ml TNF-α is diluted by 1x DBPS and    100 µl of this solution is added to each well of the ELISA    plate, followed by incubation at 4°C overnight. 2. After    overnight, perform three washes with PBST, utilizing 300    microliters for each wash 3. Position    the board inverted on the paper to ensure complete absorption of    any residual moisture 4. Block:    PBS containing 1% BSA and 0.1% Tween 20 (no Tween 20 was added    today), incubate at room temperature for one hour, gently mixing    with a decolorizing shaker. 5. Wash    with PBST for one time 6. Add    sample and primary antibody: incubate at room temperature for 2    hours     1. Positive       group：100ul/well,       2.5ug of TNFRSF1A-His protein    2. Negative       group: 100ul/well PBS    3. Experimental       group：    4. 16°C       Antibody Supernatant (A-sb, B-sb, A-LB, B-LB), diluted in 5       gradients (0, 2×, 4×, 8×, 16×)    5. 37°C       Antibody Supernatant (A-sb, B-sb, A-LB, B-LB), diluted in 5       gradients (0, 2×, 4×, 8×, 16×) 7. Perform    three washes with PBST, utilizing 300 microliters for each wash 8. Add    detection antibody：Anti-His-HRP    antibody diluted at a ratio of 1:2000, 10 mL of blocking solution    (PBS + 1% BSA + 0.1% Tween 20) diluted, incubated at 4°C    overnight 9. Perform    three washes with PBST, utilizing 300 microliters for each wash 10. Add     100ul of TMB substrate to each well. 11. Add     the stop solution to each well at a volume of 100ul. 12. Measure     the absorbance at OD450 utilizing Thermofisher MultiScan     microplate reader |

Results

![](https://static.igem.wiki/teams/5067/note1/9-1.png)
![](https://static.igem.wiki/teams/5067/note1/9-2.png)
![](https://static.igem.wiki/teams/5067/note1/9-3.png)

Notes for 2024/8/4~8/11

Overview

This file contains all record of experiment from 2024/8/4 to 2024/8/11

Experiment goals

ELISA2: molecular function test of nanoAb block the TNFA-TNFR

Participants

Kaitlyn, Hellen, Friedman, Steven, Eric, Amie, John, Sylve, Grayson, Kevin, DJL

Summary

* Standard
  curve of the ELISA kit to detect TNFa
* Prepare
  the ELISA TNFR coated plate
* ELISA
  test to verify the effect of nanobody antibody A/B supernatant to
  block TNFa binding to TNFR

Experiment process

**Standard curve of TNFa ELISA kit**  Experimental steps:  1. Prepare 1x wash solution by diluting 20x wash solution with distilled water at a ratio of 2:38.  2. Prepare 1x assay buffer by diluting 10x buffer with distilled water at a ratio of 1:10.  3. Prepare the detection antibody working solution by diluting 1x antibody working solution with distilled water at a ratio of 1:10.  4. Dilute the sample with 200 µl of 1x assay buffer.  5. Centrifuge at 10,000g for 1 minute and let it stand for 10 minutes.  6. Take 230 µl of reconstituted standard, add 230 µl of standard diluent, and perform a 1:1 dilution using the high concentration standard.  7. Take 230 µl of reconstituted standard, add 230 µl of cell culture medium, and perform a 1:1 dilution using the high concentration standard.  8. Detach any unnecessary strips from the plate and reseal them to avoid contact with the inner surface of the microwells.  9. Add 300 µl of 1x wash solution to each well on the ELISA plate and let it soak for 30 seconds.  10. Discard the wash solution and blot the microwell plate dry on absorbent paper. After washing, do not let the microwell plate dry out.  11. Add 100 µl of 2-fold serially diluted standard to the standard wells.  12. Add 50 µl of 1x assay buffer and 50 µl of the sample to the sample wells.  13. Add 100 µl of cell culture supernatant to the sample wells.  14. Add detection antibody: Add 50 µl of detection antibody working solution to each well to be tested.  15. Incubate at 100-300 rpm at room temperature (25°C ± 3°C) for 2 hours.  16. Discard the liquid, add 300 µl of wash solution to each well, and wash the plate 6 times. Blot the plate dry on absorbent paper, ensuring all residual liquid is removed.  17. Add enzyme: Add 100 µl of streptavidin working solution to each well.  18. For non-acidic liquid waste, add 1.0% sodium hypochlorite and soak for 30 minutes. For acidic liquid waste, neutralize it first, then add sodium hypochlorite.  19. Incubate: Seal the plate with a new adhesive cover. Incubate at 100-300 rpm at room temperature (25°C ± 3°C).  20. Repeat Step 16.  21. Add 100 µl of the substrate solution to each well, incubate in the dark at room temperature (25°C ± 3°C) for 5-30 minutes.  22. Add 100 µl of stop solution to each well; the color will change from blue to yellow. |

**ELISA assay****：****TNFR
coated plate and verification of the blocking effect**

1. A
   final concentration of 100 ng/ml (10ng/well) TNFR is diluted by
   1x DBPS and 100 µl of this solution is added to each well of the
   ELISA plate, followed by incubation at 4°C overnight.
2. After
   overnight, perform three washes with PBST, utilizing 300
   microliters for each wash
3. Position
   the board inverted on the paper to ensure complete absorption of
   any residual moisture
4. Block:
   PBS containing 1% BSA and 0.1% Tween 20 (no Tween 20 was added
   today), incubate at room temperature for one hour, gently mixing
   with a decolorizing shaker.
5. Wash
   with PBST for two times
6. Pre-treatment：Add
   nanoAb supernatant, Positive Ab and PBS with different
   concentration of TNFa at room temperature for one hour

   1. TNFa
      concentration: 0.25ng/ml, 1ng/ml, 2ng/ml, 4ng/ml, and 100ul/well
   2. Positive
      group：Positive-Ab(1:10000)
      + TNFa
   3. Negative
      group: PBS+TNFa
   4. Experimental
      group：
   5. 16°C
      Antibody Supernatant (A-sb, B-sb, A-LB, B-LB) + TNFa
   6. 37°C
      Antibody Supernatant (A-sb, B-sb, A-LB, B-LB) + TNFa
7. Add
   the mixture of step 18 to the coated plate and incubate at room
   temperature for 2 hours
8. Perform
   three washes with PBST, utilizing 300 microliters for each wash
9. Add
   detection antibody：Anti-TNFa-biotin
   antibody diluted at a ratio of 1:1000, 10 mL of blocking solution
   (PBS + 1% BSA + 0.1% Tween 20) diluted, incubated at room
   temperature for 1 hour
10. Add
    SA-HRP (1:250) and the final concentration is 2ug/ml, incubate at
    room temperature for 1 hour
11. Perform
    three washes with PBST, utilizing 300 microliters for each wash
12. Add
    100ul of TMB substrate to each well.
13. Add
    the stop solution to each well at a volume of 100ul.

Measure
the absorbance at OD450 utilizing Thermofisher MultiScan
microplate reader

Results

![](https://static.igem.wiki/teams/5067/note1/10-1.png)
![](https://static.igem.wiki/teams/5067/note1/10-2.png)
![](https://static.igem.wiki/teams/5067/note1/10-3.png)

Notes for 2024/8/11~8/20

Overview

This file contains all record of experiment from 2024/8/11 to 2024/8/20

Experiment goals

ELISA3: cell function test of nanoAb block the TNFa stimulation of T cell and monocyte cell

Participants

Kaitlyn, Hellen, Friedman, Steven, Eric, Amie, John, Sylve, Grayson, Kevin, DJL

Summary

* Cell
  isolation and culture
* TNFa
  stimulate the cell and verify the protect effect of nanobody
  antibody A/B by cell viability assay

Experiment process

Experimental steps:  1. Add 100μl of cells (30,000 cells) (U937 and mouse primary CD8-positive inguinal lymph node T cells) to a 96-well plate and incubate it overnight in a cell incubator.  2. Create a control group by adding TNFα+PBS. Create an experimental group by adding TNFα and 5 different antibodies, each with three concentration gradients, with the concentration halved sequentially. Then incubate the 96-well plate at 37°C for 6 hours.  3. Prepare CellTiter-Lumi solution and bring it to room temperature.  4. Take out the 96-well plate and balance it to room temperature.  5. Add 100μl of CellTiter-Lumi solution to each well, cover the plate, oscillate for 2 minutes, and incubate for 20 minutes.  6. Measure chemiluminescence using a microplate reader. |

Results

![](https://static.igem.wiki/teams/5067/note1/11.png)

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ENGINEERING
![](https://static.igem.wiki/teams/5067/s5/99-1.png)

[Overview](#nav1)

[Cycle 1: BBa\_K5067002 (p15A-Ozoralizumab )](#nav2)

[Cycle 2: BBa\_K5067003( p15A-V565)](#nav3)

[Cycle 3: Test](#nav4)

[Cycle 4: Learn](#nav5)

Overview

In recent years, targeted drugs represented by monoclonal antibodies have greatly improved the clinical treatment effect of inflammatory bowel disease, but there are unmet needs such as high prices, unresponsiveness of some patients, infection caused by long-term medication, and poor patient compliance. In this project, EcN will be genetically engineered: the nucleotide sequences of anti-TNFα nanobodies (A) will be synthesized, with both ends of Ozoralizumab(A) and nanobody-Ab V565 (B) modified to include a 6×His Tag for subsequent purification and characterization. The N-terminus of A is appended with a signaling peptide to enhance the secretion of the nanobody into the extracellular space(Figure 1). The synthesized sequences are then cloned into a transformation vector and introduced into EcN1917 through transformation. Clindamycin-resistant colonies are selected to ensure stable integration of the edited EcN1917 strain. Select multiple clones for further characterization.

![](https://static.igem.wiki/teams/5067/eng123/1.png)

Figure 1: Summary of the plasmid construction and engineered EcN 1917 bacterial

The subsequent characterization encompasses the following steps:

Cycle 1: p15A-Ozoralizumab Design and Build

Cycle 2: p15A-V565 Design and Build

Cycle 3: Test

Test1: Enzyme-linked immunosorbent assay(ELISA1) and Western Blot (WB)

Test2: ELISA2 and cell function test,

Test3: Improved verification from molecular level to cell level

Cycle 4: learn

Cycle 1: BBa\_K5067002 (p15A-Ozoralizumab )

###### Design：

Initially, we chose to perform the in-frame assembly of the plasmid in DH5α cells, followed by the transformation of the successfully assembled plasmid into EcN1917 cells. The gene sequences for both the A and B antibodies were synthesized using gene synthesis techniques. To start, we constructed the nanoAb-A plasmid through homologous recombination.(Figure 2).

![](https://static.igem.wiki/teams/5067/eng123/2.png)

Figure 2: Snapgene Plasmid and sequence diagram of nano-Ab A

###### Build:

We obtained the bacterial colony through solid plate culture overnight and we subsequently dispatched them to a biotechnology firm for sequencing (Figure 3 A ).
The results in the following two images were obtained (Figure 3 B ). Since they are solid lines, there were no base mismatch for gene mutations, indicating the success of the construction work.

![](https://static.igem.wiki/teams/5067/eng123/3.png)

Figure 3: Colony culture of DH5α-p15A-A and Sanger sequencing result.

This image depicts a culture dish in which the target genes A were successfully transformed into EcN 1917. The bacteria labeled with -1 on the left are diluted by a factor of 100, whereas those on the right remain undiluted. Figure 4 illustrates that the colony count of p15A-A bacteria significantly decreased upon 100-fold dilution, while an increased number of colonies was observed in the undiluted sample.

On the right side is the validation of monoclonal colonies. According to the image, the length is between 800-1200bp, with a specific length of 1088bp. The length is consistent with the target gene, indicating successful transformation.

![](https://static.igem.wiki/teams/5067/eng123/4.png)

Figure 4: Colony culture of transformed EcN1917 and colony PCR verification

Cycle 2: BBa\_K5067003( p15A-V565)

###### Design：

Building on the findings from cycle 1, we utilized the synthesized DNA sequence of the B antibody to develop and validate the nanoAb-B plasmid. This involved constructing the plasmid and confirming its functionality through a series of validation steps.(Figure 5).

![](https://static.igem.wiki/teams/5067/eng123/5.png)

Figure 5: Snapgene diagram of p15A-nanoAb-B plasmid

###### Build:

We obtained the bacterial colony through solid plate culture overnight and we subsequently dispatched them to a biotechnology firm for sequencing (Figure 6A ).
The results in the following two images were obtained (Figure 6B ). Since they are solid lines, there were no base mismatch for gene mutations, indicating the success of the construction work.

![](https://static.igem.wiki/teams/5067/eng123/6.png)

Figure 6: Colony culture of DH5α-p15A-B and Sanger sequencing result.

Figure 7 illustrates that the colony count of p15A-B bacteria significantly decreased upon 100-fold dilution, while an increased number of colonies was observed in the undiluted sample. In contrast, for the p15A-B gene subjected to 100-fold dilution, there was virtually no observable growth of colonies. On the right side is the validation of monoclonal colonies. According to the image, the length is between 800-1200bp, with a specific length of 1088bp. The length is consistent with the target gene, indicating successful transformation.

![](https://static.igem.wiki/teams/5067/eng123/7.png)

Figure 7: Transformation of the p15A-nanoAb-B plasmid to EcN1917 and colony PCR verification

Cycle 3: Test

###### Test 1: expression verification by WB and ELISA (TNFα-coated plate)

We successfully constructed the EcN1917-nanoAb-A and EcN1917-nanoAb-B strains, which were cultured at 16°C and 37°C, respectively, in LB and SB media for a duration of 48 hours. Following centrifugation and subsequent filtration through a 0.22 μm membrane to eliminate bacterial cells, we obtained the final antibody product(Figure 8). The expression of the nanoAb-His fusion protein was effectively detected via Western blotting. We coated the assay plate with TNFα and employed an Anti-His-HRP ELISA to detect the antibody, yielding a significant light signal. By integrating these two experimental approaches, we preliminarily conclude that the supernatant from the engineered probiotic culture contains nanobody proteins, with nanobody B exhibiting higher expression levels than nanobody A.

![](https://static.igem.wiki/teams/5067/eng123/8-1.png)
![](https://static.igem.wiki/teams/5067/eng123/8-2.png)

Figure 8: Expression verification of nanoAb-A and nanoAb-B by Western blot and ELISA (coated with TNFα)

Note: The PBS is the blank control group and the Posi-Ab means positive control group. AL means desired gene A in LB medium, AS means gene A in SB medium at 37 °C, BL means gene B in LB medium at 37 °C, BS means gene B in SB medium at 37 °C, AL16 means gene A in LB medium A at 16 °C, AS16 means gene A in SB medium at 16 °C, BL16 means gene B in LB medium at 16 °C, BS16 means gene B in SB medium at 16 °C.

###### Test 2: ELISA2 to investigate the blocking effect from molecular level of TNFα and TNFR

Using the nanobody supernatant collected in Test 2, we coated TNFR onto the plate and measured the quantity and affinity of TNFα bound to it using an Anti-TNFα-HRP detection antibody. The positive control group consisted of a commercially available anti-TNFα antibody, while the experimental group included eight different nanobody supernatants. PBS served as the negative control. We then evaluated the blocking efficacy at varying TNFα concentrations. Notably, the AL, AL16, and AS16 groups demonstrated significant blocking capabilities at both 0.5 ng/mL and 1 ng/mL TNFα concentrations(Figure 9).

![](https://static.igem.wiki/teams/5067/eng123/9.png)

Figure 9: ELISA (coated wit TNFR) verify the blocking effect of nanoAb on the binding of TNFα to TNFR.

Note:The PBS is the blank control group and the Posi-Ab means positive control group. AL means desired gene A in LB medium, AS means gene A in SB medium at 37 °C, BL means gene B in LB medium at 37 °C, BS means gene B in SB medium at 37 °C, AL16 means gene A in LB medium A at 16 °C, AS16 means gene A in SB medium at 16 °C, BL16 means gene B in LB medium at 16 °C, BS16 means gene B in SB medium at 16 °C.

###### Test 3: Cell viability test to verify the protect effect of nanobody antibody on cell

After TNF-a binds to the TNF receptor (TNFR) on the cell surface, it initiates apoptosis and necrosis programs, ultimately leading to cell death. However, after TNFα binds to the nanoantibody, it can no longer bind to TNFR, and therefore cannot cause cell death. Therefore, we further studied the effect of nanoantibodies in inhibiting inflammatory factors through cell experiments.

TNF-α was mixed with a series of nanoantibodies at different concentrations (a total of 5 nanoantibodies were obtained by culture, and the concentration of each nanoantibody was reduced by half in turn, and the mixture of TNF-α and PBS was used as a negative control) and incubated with U937 cells for 6 hours. The viability of each group of cells was then detected using the CellTiter-Lumi kit. The results (Figure 10) showed that compared with the control group, the nanoantibodies obtained by the 5 culture methods all showed a good effect in inhibiting cell death. And this effect is positively correlated with the concentration of the nanoantibody, that is, as the concentration increases, the effect of inhibiting cell death increases.

![](https://static.igem.wiki/teams/5067/eng123/10.png)

Figure 10: Cell viability test by Cell-titer assay to verify the protect effect of nanoAb on cell stimulated by TNFα.

Cycle 4: Learn

1. The expression level of Ozoralizumab (

A ) can be further improved during the experiment, and WB experiment is a little fuzzy, we can run a better WB diagram. At the same time, ELISA experiments can be done more gradient protein concentration verification.

2. In the future, the iGEM team can further explore and optimize p15-B, for example, the B-carrier protein expression is not good, which requires more experiments to optimize it.

3. Other iGEM teams can also further optimize the dual-target nanobodybased on our project.They can use nanobodies in other fields.

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EXPERIMENTS
![](https://static.igem.wiki/teams/5067/s5/99-1.png)

[Outline](#nav1)

[Apparatus & Materials](#nav2)

[Procedure](#nav3)

[References and sources](#nav4)

Outline

![](https://static.igem.wiki/teams/5067/t1.jpg)

Apparatus & Materials

###### 1.1 Reagents:

Tryptone, yeast extract, sodium chloride, water, Prime Mix, p15A-GEx-A/B, Primer-R, Primer-F, Template, TAE buffer, Marker, NEB dye, Wash solution, Elution buffer, Buffer SP1 / Buffer SP2 / Buffer SP3 / Buffer S / Buffer DW1 / Wash Solution, Recombinase, Mold label plate, Detection antibody, Standard diluent, Streptavidin, Detection buffer solution, Chromogenic substrate, Wash solution, LB/SB medium, A bacterial liquid, B bacterial liquid

###### 1.2 Experimental tools:

Beaker, Pipette, Medicine spoon, Weighing paper, PCR thermal cycler, Centrifuge tube, pipette, Pipette tip, Centrifuge, Electronic balance, Bain-marie, Microwave oven, UV transilluminator, Electrophoresis chamber, Thermostat, Clean bench, Automatic ice maker.

Procedure

###### 2.1Disposition of LB Culture Base

Goal: To complete the LB culture medium with 500ml water, which can be used for pre-culturing bacterial strains in experiments, to multiply the bacterial strains and meet the requirements for use. It can also be used for culturing Escherichia coli.
Experimental steps:

1 .Weigh using an electronic balance and a medicine spoon, paying attention to the weight of the weighing paper and keeping the balance closed.

2. Pour the weighed solid into a conical flask

3. Constant volume: Measure 500ml of water with a measuring cylinder

4. Mix the measured solid and water together

5. Finally, plug the prepared liquid culture medium and keep it closed and flowing

6. Sterilize, if not possible, temporarily store in the refrigerator.

###### 2.2 Construction of p15A-GEx-A/B

###### 2.2.1 Obtaining target gene fragments by PCR

Goal: PCR (polymerase chain reaction), which rapidly replicates DNA through a very simple method, can rapidly increase the quantity of trace amounts of DNA in a short period of time. PCR has a great role in medical research and forensic fields. During DNA replication, the two strands of the parent DNA are used as templates to synthesize two new strands based on the principle of complementary bases, resulting in the formation of DNA with the same sequence of bases as the parent. PCR is the process of denaturing DNA in a high-temperature environment, allowing it to unwind. Then cool down and allow the primer (containing approximately 20 bases) to bind to complementary DNA. DNA without a primer does not bind. Finally, heat up and extend. Taq enzyme pairs with the primer and synthesizes it into offspring DNA, and then repeats this process.

Experimental steps:

1. Adjust the numbers with a pipette first, and then mix the materials

2. Inactivate at a high temperature of 98 °C Celsius, causing DNA to change from double stranded to single stranded

3. Deactivate at 56 °C and primer binding to DNA

4. 72 °C extended DNA pairing

5. Repeat about 30 times to obtain a large amount of DNA

###### 2.2.2 Agarose gel construction and deployment

1. Dissolve 0.3g Agarose in 30mL Erlenmeyer flask

2. Add TAE buffer (Running buffer)

3. Heat the material with microwave

4. Melt the mixture at 30 sec interval

5. See whether bubbles are forming, if did successfully mixed

6. Add 3μl nucleic acid gel stain (10000x)

7. Let it cool for 5~10 minutes

8. Pour the agarose mixture into the casting tray

9. Place the appropriate well comb to create the wells

10. Wait to solidify 7~10 minutes

11. Remove the comb and place the gel in the gel box

###### 2.2.3 Agarose gel electrophoresis

Purpose: To separate, identify, and purify DNA fragments.

Experimental steps:

1. Prepare the gel: Mix 200 ml of TAE buffer with 2 g of agarose, and heat in a microwave for 5 minutes.

2. Wait for the solution to cool until it is no longer hot to the touch, then add NEB dye at a volume of 1/10,000 of the solution.

3. Pour the solution into the gel tray.

4. Allow the gel to solidify (approximately 33 minutes), then use a pipette to add PCR products and marker to each well.

5. Perform electrophoresis at 180 V for 20 minutes.

6. Capture the image under UV light

###### 2.2.4 Recovery of DNA

Purpose: Recover DNA and determine its concentration

Experimental steps:

1. Cut the gel and place it into a tube.
2. Add gel dissolution solution (3 times the volume of the gel weight, like 100ml Buffer for 0.1g gel).
3. Heat at 50°C in a water bath for 10 minutes, shaking continuously.
4. Transfer 700µl of the solution to the combined adsorption column and collection tube.
5. Centrifuge to separate DNA at 12,000 rpm for 1 minute.
6. Repeat this step until all DNA is filtered through the binding column.
7. Add 600µl of wash solution and centrifuge at 12,000 rpm for 1 minute. Discard the waste liquid and place the adsorption column into a new collection tube. Repeat this washing step once.
8. Heat the elution buffer at 65°C for 2 minutes. Then, suspend and add the elution buffer to the adsorption column and a new centrifuge tube. Centrifuge at 12,000 rpm for 1 minute.
9. Measure the concentration: first measure the water, then measure the elution buffer separately. Record the A260 and concentration data.

###### 2.3 Transformation of plasmid into E. coli

2.3.1 Homologous Recombination

Purpose: ligate target DNA fragment with vector backbone fragment

Experimental steps:

1. Transfer the DNA recovery product to a PCR tube.
2. Add 5µl of recombinase to each tube.
3. Heat at 50°C in a PCR machine for 30 minutes.
4. Store the finished product at -20°C.

###### 2.3.2 Transformation of p15A-GEx-A/B into E. coli DH5α via heat-shock

Purpose: Cultivate E. coli

Experimental steps:

1. Take out E. coli DH5a competent cells from -80 °C refrigerator;

2. Add 10μl of p15A-GEx-A/B and 50μl of DH5α cells into an EP tube.

3. Mix the EP tubes with the mini-centrifuge for 5 seconds.

4. Put the EP tubes on ice for 30 minutes.

5. Heat shock the samples at 42 °C for 45 seconds in the water bath.

6. Put the EP tubes in ice for 5 minutes.

7. Transfer the bacteria samples from the EP tubes into liquid LB medium. This step is performed in the clean bench.

8. Shake the cultures for 2 hours at 37 °C, at 220rpm.

9. Spread coat 50μl on the petri dishes with LB solid culture medium containing Chloramphenicol, respectively.

10. Inverted culture at 37 °C and/or 16 °C for 12-16 hours.

###### 2.3.3 Plasmid Extraction

purpose: To isolate plasmid DNA from bacteria for use as a vector.

Experimental
steps:

1. Check if RNase A has been added to Buffer SP1. Verify if absolute
ethanol has been added to the Wash Solution. Check for any precipitation in Buffer SP2 and SP3.

2. Column Equilibration: Add 500 µL of Buffer S to the column (place the
column in a collection tube), centrifuge at 12,000 g for 1 minute, discard the waste liquid from the collection
tube, and place the column back in the collection tube.

3. Take 1.5-5 mL of overnight bacterial culture, centrifuge at 8,000 g for
2 minutes to collect the cells, and discard the culture medium.

4. Add 250 µL of Buffer SP1 to the pellet and resuspend the cells
thoroughly.

5. Add 250 µL of Buffer SP2, gently invert the tube 5-10 times to mix, and
let it sit at room temperature for 2-4 minutes.

6. Add 350 µL of Buffer SP3, gently invert the tube 5-10 times to
mix.

7. Centrifuge at 12,000 x g for 5-10 minutes. Transfer the supernatant to
the column, centrifuge at 8,000 g for 30 seconds, and discard the liquid from the collection tube.

8. Add 500 µL of Wash Solution, centrifuge at 9,000 g for 30 seconds, and
discard the liquid from the collection tube.

9. Repeat Step 8 once.

10. Spin the empty column at 9,000 x g for 1 minute.

11. Place the column into a clean 1.5 mL centrifuge tube, add 50-100 µL of
buffer.

12. Elution Buffer to the center of the membrane, let it sit at room
temperature for 1 minute, and then centrifuge for 1 minute. Save the DNA solution in the tube.

13. Test concentration (range 100 to 200).

2.3.4 Monoclonal verification

Purpose: To generate a specific type of antibody to recognize and bind to
a particular antigen. By cloning a single B cell, a large quantity of identical antibodies can be produced,
which is very useful in research, diagnosis, and treatment.

Experimental
steps:

1. Prepare the gel: Mix 200 ml of TAE buffer with 2 g of agarose, and heat in a microwave for five
   minutes.
2. Wait
   for the solution to cool until it is no longer hot to the touch, then add NEB dye at a volume of 1/10,000 of
   the solution.
3. Pour
   the solution into the gel tray.
4. Allow the gel to solidify (approximately 33 minutes), then use a pipette to add PCR products and
   marker to each well.
5. Perform electrophoresis at 180 V for 20 minutes.
6. Capture the image.

2.3.5 Amplification culture of engineered Escherichia coli

Experimental
steps:

1. Add 200μL of bacterial liquid to each well of the microplate.

2. Measure the OD.

3. Dilute the four bacterial liquids to the same OD value.

4. Add 30 mL of culture medium.

5. Place in a shaker at temperatures of 37 °C and 16 °C.

2.3.6 Transformation of p15A-GEx-A/B into EcN 1917 via heat-shock

Experimental
steps:

1. Take out EcN competent cells from -80 °C refrigerator;

2. Add 10μl of p15A-GEx-A/B and 50μl of EcN into an EP tube.

3. Mix the EP tubes with the mini-centrifuge for 5 seconds.

4. Put the EP tubes on ice for 30 minutes.

5. Heat shock the samples at 42 °C for 45 seconds in
the water bath.

6. Put the EP tubes in ice for 5 minutes.

7. Transfer the bacteria samples from the EP tubes into liquid LB medium.
This step is performed in the clean bench.

8. Shake the cultures for 2 hours at 37 °C, at 220rpm.

9. Spread coat 50μl on the petri dishes with LB solid culture medium
containing Chloramphenicol, respectively.

10. Inverted culture at 37 °C and for 12-16
hours.

2.3.7 Extraction plasmid from EcN

Goal: (1) Dissolve bacteria (2) Isolate DNA (3) Purification

Check if the buffer has RNase A

Check anhydrous ethanol/precipitate

Experimental
steps:

1. Pour
   out the supernatant, add it to the adsorption column, add 500 microliters of buffer, centrifuge for 1 minute,
   discard the waste liquid, and then pour it back.
2. Mix
   well and centrifuge 1.5-5ml for 2 minutes
3. Add
   250 microliters of buffers to the precipitate
4. Centrifuge and add 250 microliters of Buffer SP2. Slowly invert 5-10 times and let it stand for 2-4
   minutes
5. Add
   350 microliters of buffers and invert 10 times
6. Centrifuge for 5-10 minutes, transfer the supernatant to the adsorption column and centrifuge for
   30 seconds
7. Add
   500 microliters of WASH SOLUTION and centrifuge for 30 seconds, repeat once
8. Transfer the centrifugal adsorption column into a centrifuge tube and add ELUTION BUFFER.
   Centrifuge and let it stand for 1 minute
9. The
   concentration data is around 100-200.

2.4 Validation of nano-antibody expression by Western Blot

Step 1:
Sample Preparation

Prepare protein lysates: Ensure that your lysates contain a reducing
agent like DTT or β-mercaptoethanol.

Boil samples: Boil the lysates at 95°C for 5 minutes to denature the
proteins and allow them to bind to the SDS.

Step 2:
Electrophoresis (SDS-PAGE)

Load samples: Load equal amounts of protein
lysates into the wells of the pre-cast polyacrylamide gel.

Run the gel: Apply a
constant voltage (e.g., 80V for stacking gel, then increase to 120V for resolving gel) until the tracking dye
reaches the bottom of the gel.

Step 3: Transfer (Blotting)

Assemble transfer sandwich: Place the
nitrocellulose or PVDF membrane on top of the gel, ensuring there are no air bubbles.

Perform transfer: Use
either a semi-dry transfer apparatus or a wet transfer tank. Apply a constant current (e.g., 300mA for 90
minutes) to transfer the proteins from the gel to the membrane.

Step 4: Blocking

Block non-specific binding sites: Incubate the membrane in blocking buffer
for 1 hour at room temperature to prevent non-specific antibody binding.

Step 5: Primary Antibody Incubation

Prepare primary antibody: Dilute the anti-His tag antibody in blocking
buffer according to the manufacturer's instructions (1:2000).

Incubate: Incubate the membrane with the diluted primary antibody
overnight at 4°C.

Step 6: Wash

Wash the membrane: Wash the membrane three times
with TBS-T (Tris-buffered saline with Tween-20) to remove unbound primary antibody.

Step 7:
Secondary Antibody Incubation

Prepare secondary antibody: Dilute the HRP-conjugated secondary antibody
in blocking buffer.

Incubate: Incubate the membrane with the diluted secondary antibody for 1
hour at room temperature.

Step 8: Final Wash

Wash the membrane: Wash the membrane three times with TBS-T followed by a
final wash with TBS alone.

Step 9: Detection

Apply ECL substrate: Apply the chemiluminescent
substrate to the membrane.

Develop the signal: Place the membrane in a cassette and expose it to a
digital imaging system to visualize the bands.

2.5 Validation of nano-antibody expression by ELISA

Experimental
steps:

1. Prepare 1x wash solution by diluting 20x wash solution with distilled
water at a ratio of 2:38.

2. Prepare 1x assay buffer by diluting 10x buffer with distilled water at
a ratio of 1:10.

3. Prepare the detection antibody working solution by diluting 1x antibody
working solution with distilled water at a ratio of 1:10.

4. Dilute the sample with 200 µl of 1x assay buffer.

5. Centrifuge at 10,000g for 1 minute and let it stand for 10
minutes.

6. Take 230 µl of reconstituted standard, add 230 µl of standard diluent,
and perform a 1:1 dilution using the high concentration standard.

7. Take 230 µl of reconstituted standard, add 230 µl of cell culture
medium, and perform a 1:1 dilution using the high concentration standard.

8. Detach any unnecessary strips from the plate and reseal them to avoid
contact with the inner surface of the microwells.

9. Add 300 µl of 1x wash solution to each well on the ELISA plate and let
it soak for 30 seconds.

10. Discard the wash solution and blot the microwell plate dry on
absorbent paper. After washing, do not let the microwell plate dry out.

11. Add 100 µl of 2-fold serially diluted standard to the standard
wells.

12. Add 50 µl of 1x assay buffer and 50 µl of the sample to the sample
wells.

13. Add 100 µl of cell culture supernatant to the sample wells.

14. Add detection antibody: Add 50 µl of detection antibody working
solution to each well to be tested.

15. Incubate at 100-300 rpm at room temperature (25°C ± 3°C) for 2
hours.

16. Discard the liquid, add 300 µl of wash solution to each well, and wash
the plate 6 times. Blot the plate dry on absorbent paper, ensuring all residual liquid is removed.

17. Add enzyme: Add 100 µl of streptavidin working solution to each
well.

18. For non-acidic liquid waste, add 1.0% sodium hypochlorite and soak for
30 minutes. For acidic liquid waste, neutralize it first, then add sodium hypochlorite.

19. Incubate: Seal the plate with a new adhesive cover. Incubate at
100-300 rpm at room temperature (25°C ± 3°C).

20. Repeat Step 16.

21. Add 100 µl of the substrate solution to each well, incubate in the
dark at room temperature (25°C ± 3°C) for 5-30 minutes.

22. Add 100 µl of stop solution to each well; the color will change from
blue to yellow.

2.6 Validation of nano-antibody function

Experimental
steps:

1. Add 100μl of cells (30,000 cells) (U937 and mouse primary CD8-positive
inguinal lymph node T cells) to a 96-well plate and incubate it overnight in a cell incubator.

2. Create a control group by adding TNFα+PBS. Create an experimental group
by adding TNFα and 5 different antibodies, each with three concentration gradients, with the concentration
halved sequentially. Then incubate the 96-well plate at 37°C for 6 hours.

3. Prepare CellTiter-Lumi solution and bring it to room
temperature.

4. Take out the 96-well plate and balance it to room temperature.

5. Add 100μl of CellTiter-Lumi solution to each well, cover the plate,
oscillate for 2 minutes, and incubate for 20 minutes.

6. Measure chemiluminescence using a microplate reader.

References and sources
- https://www.chem17.com/st421009/product\_36962112.html
- https://www.procell.com.cn/view/499.html

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RESULTS
![](https://static.igem.wiki/teams/5067/s5/99-1.png)

[Overview](#nav1)

[Results of our work](#nav2)

[Protein expression](#nav3)

[Function verification](#nav4)

Overview

EcN can be used as a probiotic for the treatment of intestinal related diseases due to its good ability to instantly implant into the intestine and non-pathogenicity. According to recent research, targeted drugs such as monoclonal antibodies have greatly improved the clinical efficacy of diseases such as intestinal inflammation, but there are also problems such as high prices, ineffective treatment for patients, and infections.The design of our experiment employs a comprehensive approach to treating the disease through a combination of therapeutic agents, including A-nanobody, a nanobody derived from Ozoralizumab, and B-nanobody, a nanobody associated with V565, in conjunction with Escherichia coli Nissle 1917. And we added a signal peptide to the front of the antibody, which can release the nanobody to the extracellular. The engineered EcN 1917 couldsecret the nanobody A and B, which binds to the natural TNFα and block the inflammatory reaction(Figure 1).

![](https://static.igem.wiki/teams/5067/res12/r1.png)

Figure 1: Summary of the application of engineered EcN 1917 bacterial for treatment of TNFα associated inflammatory bowel disease.

Results of our work
![](https://static.igem.wiki/teams/5067/res12/0.png)

###### 1.1 Design the PCR primer according to plasmid diagram

![](https://static.igem.wiki/teams/5067/res12/2.png)

Figure 2: The plasmid maps of p15A-A and p15A-B.

We identified the gene sequences of target gene A and target gene B, followed by the selection of the p15A vector for obtaining their plasmid profiles. Subsequently, homologous recombination was utilized to merge them( Figure 2).

###### 1.2 Amplification of target fragment and vector backbone

We utilized polymerase chain reaction (PCR) to amplify the target DNA and prepared an agarose gel for electrophoresis to separate the amplified DNA fragments. The final results were analyzed using ultraviolet imaging to visualize the DNA bands.

![](https://static.igem.wiki/teams/5067/res12/3.png)

Figure 3: PCR amplification of target A/B and p15A vector backbone

Figure 3A displays the electrophoresis image of the p15A vector, with molecular markers and corresponding values shown on the left. The lengths of p15A-1 and p15A-2 fall between 5000 bp and 7500 bp, with the p15A vector having a length of 6535 bp. The target band aligns with the expected size of the target gene.

Figure 3B presents the electrophoresis image of target gene A/B, with markers and values on the left. The length of target gene A/B ranges from 500 bp to 1000 bp, and the specific length is 622 bp. The target band is consistent with the expected size of the target gene.

To ensure that the DNA is ready for use in subsequent experiments, we removed redundant agents, substrates, and Taq polymerase through solubilization and centrifugation, ultimately isolating the target fragment.

###### 1.3 Transformation of plasmid into E. coli DH5α

###### 1.3.1 Homologous recombination

Untreated receptor cells are generally not receptive to recombinant molecules, making transformation inefficient. To address this, we employed methods to induce the cells into an optimal receptive state, significantly improving transformation efficiency. This was achieved by adjusting and mixing calcium chloride, culture medium, and regulating the temperature to prepare the cells.

For the transformation process, we mixed agar, plasmids, and other necessary reagents in specific proportions, followed by ice bath incubation and temperature regulation. The final transformation step was performed using a shaking table to ensure even mixing and proper conditions.

We first prepared the cells, bringing them into the most suitable receptive state, and then carried out the plasmid transformation. Using the nonpathogenic Escherichia coli bacterial transformation method, we successfully amplified the target plasmid DNA, providing essential raw materials for subsequent experiments.

###### 1.3.2 Transformation of p15A-GEx-A/B into E. coli DH5α via heat-shock

Preparation of competent cells: Untreated receptor cells are insensitive to recombinant molecules and difficult to achieve conversion. We use methods to induce cells to be in the most suitable receptive state with a high conversion frequency. Prepare cells by adjusting and mixing calcium chloride, culture medium, and temperature. For the transformation process, we combined the plasmid with competent cells and incubated the mixture in an ice bath for a minimum of 30 minutes. This was followed by procedures including heat shock and subsequent ice stimulation.

###### 1.3.3 Verification of monoclonal colonies

![](https://static.igem.wiki/teams/5067/res12/4.png)

Figure 4: Colony culture of DH5α-p15A-A, DH5α-p15A-B and Sanger sequencing result.

We obtained the bacterial colony through solid plate culture overnight and we subsequently dispatched them to a biotechnology firm for sequencing (Figure 4 A and C).

The results in the following two images were obtained (Figure 4 B and D). Since they are solid lines, there were no base mismatch for gene mutations, indicating the success of the construction work.

###### 1.4 Transformation of p15A-GEx-A/B into EcN 1917

![](https://static.igem.wiki/teams/5067/res12/5.png)

Figure 5: Colony culture of transformed EcN1917 and colony PCR verification

This image depicts a culture dish in which the target genes A and B were successfully transformed into EcN 1917. The bacteria labeled with -1 on the left are diluted by a factor of 100, whereas those on the right remain undiluted. Figure 5 illustrates that the colony count of p15A-A bacteria significantly decreased upon 100-fold dilution, while an increased number of colonies was observed in the undiluted sample. In contrast, for the p15A-B gene subjected to 100-fold dilution, there was virtually no observable growth of colonies.

On the right side is the validation of monoclonal colonies. According to the image, the length is between 800-1200bp, with a specific length of 1088bp. The length is consistent with the target gene, indicating successful transformation.

###### 1.5 Culture of EcN1917- A/B at LB/SB at 16/37 Celsius and collect the supernatant of medium

1. The preceding night, the bacteria were inoculated into LB liquid culture medium and incubated overnight. Chloramphenicol was subsequently added to achieve a final concentration of 25 micrograms per milliliter, specifically for Nissle1917 expressing antibodies A and B.
2. On the following evening, measure the optical density (OD) and select the strain with the highest OD value. Inoculate this strain at a concentration of 1% of its volume into SB medium containing chloramphenicol and adjust the volumes of the remaining strains to match this maximum OD value. Based on the added volume, proportionally increase the inoculation volume in LB medium while incubating at 16°C and shaking at 220 rpm for 12 hours; prior to incubation at 37°C, measure the OD to ensure that all inoculated liquid starts from an equivalent initial volume.
3. Centrifuge at 3000 g for 5 minutes at 4°C, collect the supernatant, filter it through a 0.22-micron filter, and store the resulting solution at -80°C.

Protein expression

###### 2.1 Validation of the existence and expression of nanoAb-A/B by Western Blot

![](https://static.igem.wiki/teams/5067/res12/6.png)

Figure 6: Nano-antibody expression in the supernatant verified by Western blot assay

We cultured Escherichia coli Nissle 1917 (EcN 1917) transformed with A/B plasmids and optimized the cultivation conditions at both 16°C and 37°C, using LB and SB media. After the cultivation process, we collected the supernatant, centrifuged it, and filtered out bacterial cells to obtain the final product. We then selected the antibody products A-LB and B-LB for further analysis.

To confirm the successful expression of nanoAbs, we performed Western blotting to detect the His tag. As shown in the figure, a band corresponding to the His tag appears around 15 kDa, which aligns with molecular weight predictions and the theoretical sequence of the nanoAbs. Additionally, the expression level of nanoAb-B is slightly higher compared to nanoAb-A, as indicated by the intensity of the bands in the Western blot in the Figure 6.

###### 2.2 Validation of nano-antibody expression by ELISA

![](https://static.igem.wiki/teams/5067/res12/7.png)

Figure 7: Expression of Nano-antibody A and B by ELISA(coated with TNFα).

The ELISA verified the relationship between A/B proteins and TNF-a binding ability. The PBS is the blank control group and the Posi-Ab means positive control group. AL means desired gene A in LB medium, AS means gene A in SB medium at 37 °C, BL means gene B in LB medium at 37 °C, BS means gene B in SB medium at 37 °C, AL16 means gene A in LB medium A at 16 °C, AS16 means gene A in SB medium at 16 °C, BL16 means gene B in LB medium at 16 °C, BS16 means gene B in SB medium at 16 °C.

The antibody binding signal obtained at 37°C is significantly higher than that obtained at 16°C, and the B antibody signal is higher than the A antibody signal, indicating that the expression level of antibody B is higher, which is consistent with the Western blot results shown in Figure 7. The results presented above demonstrate that the nanobodies we engineered have been successfully expressed in the supernatant of the harvested EcN1917 bacterial suspension.

![](https://static.igem.wiki/teams/5067/res12/8.png)

Figure 8: Color diagram of the enzyme plate: Add TMB for color development (left) and after termination (right).

Additionally, we established a standard curve utilizing a commercial kit for TNF-α to function as a reference standard for our detection system and its corresponding results in the Figure 8and Figure 9.

![](https://static.igem.wiki/teams/5067/res12/9.png)

Figure 9: Standard curve of ELISA.

Function verification

###### 3.1 Validation of nano-antibody function by molecular assay

![](https://static.igem.wiki/teams/5067/res12/10.jpg)

Figure 10: The ELISA verified the blocking effect of A/B proteins on the interaction of TNFα-TNFR.

Initially, we coated the ELISA plate with TNFR, followed by the addition of various antibody sera mixed with TNFα at four distinct concentrations. Finally, we quantified the resultant light signal by detecting antibodies of anti-TNFα-HRP. We find that blank control group need to higher than positive control group in the Figure 10. So, we found that only in the concentration of 1ng/ml and 0.5ng/ml TNFα, the nano-antibody A shows block effect on the binding of TNFα-TNFR.

###### 3.2 Validation of nano-antibody function by cell viability assay

![](https://static.igem.wiki/teams/5067/res12/11.png)

Figure 11: The luminescence-based cell viability assay for antibody A and B under different temperatures and growth conditions.

After TNF-α binds to the TNF receptor (TNFR) on the cell surface, it triggers apoptosis and necrosis pathways, ultimately leading to cell death. However, when TNF-α binds to a nanoantibody, it is prevented from binding to TNFR, thereby inhibiting the cell death process. To further explore the effect of nanoantibodies in inhibiting inflammatory factors, we conducted cell experiments.

TNF-α was mixed with a series of nanoantibodies at different concentrations (five nanoantibodies obtained from culture, each diluted stepwise by half), and TNF-α mixed with PBS was used as a negative control. This mixture was incubated with U937 cells for 6 hours. Cell viability in each group was then assessed using the CellTiter-Lumi kit.

The results (Figure 11) demonstrated that, compared to the control group, the nanoantibodies produced by the five culture methods exhibited strong inhibition of cell death. Furthermore, this inhibitory effect was positively correlated with the concentration of the nanoantibody—meaning that as the concentration of the nanoantibody increased, the ability to prevent cell death also improved.

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