iGEM 2025 — Team beijing-hs

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Main Timeline",
"content": '

Each month is depicted with a grid system where each
grid corresponds to a 3-day period. The shaded regions indicate the days designated for work.

January：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |  |

February：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Establishment of the project team |  |  |  |  |  |  |  |  |  |  |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |  |

March：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Establishment of the project team |  |  |  |  |  |  |  |  |  |  |
| Development of Project Plan |  |  |  |  |  |  |  |  |  |  |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |  |

April：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Establishment of the project team |  |  |  |  |  |  |  |  |  |  |
| Development of Project Plan |  |  |  |  |  |  |  |  |  |  |

May：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Subject determination |  |  |  |  |  |  |  |  |  |  |
| Theoretical study |  |  |  |  |  |  |  |  |  |  |
| Expert Interview Guide |  |  |  |  |  |  |  |  |  |  |

June：

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Theoretical study |  |  |  |  |  |  |  |  |  |  |
| Subject determination |  |  |  |  |  |  |  |  |  |  |
| Experimental design |  |  |  |  |  |  |  |  |  |  |
| Safety training |  |  |  |  |  |  |  |  |  |  |

July:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Experimental design |  |  |  |  |  |  |  |  |  |  |
| Problems discussion |  |  |  |  |  |  |  |  |  |  |
| Record experiment report |  |  |  |  |  |  |  |  |  |  |
| Building the Team Wiki |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |
| Implement experimental plan |  |  |  |  |  |  |  |  |  |  |
| Expert Interview Guide |  |  |  |  |  |  |  |  |  |  |

August:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Implement experimental plan |  |  |  |  |  |  |  |  |  |  |
| Problems discussion |  |  |  |  |  |  |  |  |  |  |
| Analysis of experimental results |  |  |  |  |  |  |  |  |  |  |
| Building the Team Wiki |  |  |  |  |  |  |  |  |  |  |

September:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Problems discussion |  |  |  |  |  |  |  |  |  |  |
| Building the Team Wiki |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |
| Preparation of materials |  |  |  |  |  |  |  |  |  |  |
| Analysis of experimental results |  |  |  |  |  |  |  |  |  |  |

October:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Preparation of materials |  |  |  |  |  |  |  |  |  |  |
| Project review |  |  |  |  |  |  |  |  |  |  |
| Building the Team Wiki |  |  |  |  |  |  |  |  |  |  |
| Project Defense Preparation |  |  |  |  |  |  |  |  |  |  |

'
},{
"title": "Record of Main Experimental Content",
"content": '

**Pre-experiment safety training：**

**The main content is as follows：**

I. Always wear required PPE: lab coat, gloves, and
safety goggles in the laboratory.

II. Know locations and operations of emergency
equipment (eyewash stations, fire extinguishers, safety showers).

III. Never consume food/beverages or apply cosmetics
in lab areas.

IV. Follow proper handling/disposal protocols for
chemicals, biohazards, and sharps.

V. Maintain clean, organized workspaces to prevent
accidents and contamination.

VI. Immediately report spills, accidents, or injuries
to the supervisor.

VII. Perform only authorized, trained procedures
under supervision.

VIII. Operate equipment strictly per designated
functions and safety protocols.

IX. Memorize emergency procedures including
evacuation routes and assembly points.

X. Attend regular safety training and comply with all
guidelines to ensure workplace safety.

**Part 1: Plasmid Extraction**

**Purpose**

To extract and purify plasmid DNA from cultured
bacteria for use in downstream applications such as PCR.

**Procedure**

1. **Bacterial Collection**
   * Transfer 1.5 mL of bacterial culture into a centrifuge tube.
   * Centrifuge at 10,000 × g for 1 minute at room temperature.
   * Discard the supernatant.
2. **Cell Resuspension**
   * Add 250 μL of Solution A.
   * Resuspend the pellet completely.
3. **Cell Lysis**
   * Add 250 μL of Solution B.
   * Gently invert 4–6 times.
   * Let stand for 2 minutes to lyse cells.
4. **Protein and Debris Precipitation**
   * Add 350 μL of Solution C.
   * Gently invert until white precipitates form.
   * Centrifuge at 10,000 × g for 10 minutes at room temperature.
5. **DNA Binding**
   * Transfer the supernatant to a new tube containing the DNA adsorption column.
   * Centrifuge at 10,000 × g for 1 minute.
6. **Protein Removal**
   * Discard the filtrate.
   * Add 500 μL Buffer HB.
   * Centrifuge for 1 minute at 10,000 × g.
7. **Column Washing (x2)**
   * Add 750 μL Wash Buffer, centrifuge for 1 minute.
   * Discard filtrate.
   * Repeat with another 750 μL Wash Buffer and centrifuge again.
8. **Column Drying**
   * Centrifuge the empty column at 10,000 × g for 1 minute to remove ethanol.
9. **DNA Elution**
   * Place column into a clean tube.
   * Add 50 μL TE Buffer to the center of the membrane.
   * Let sit 2–5 minutes.
   * Centrifuge at 10,000 × g for 1–5 minutes.
   * Store eluted DNA at –20°C.
10. **DNA Quantification**

* Measure concentration and purity of eluted plasmid using a Nanodrop.

![0e552f927cf9974013da62513dc55e2c](https://static.igem.wiki/teams/5734/notebook/1.webp)![2f52b135a400eac1e86549a9c4042ab1](https://static.igem.wiki/teams/5734/notebook/2.webp)

**Part 2: Agarose Gel Electrophoresis, Gel
Extraction, Seamless Cloning, and Transformation**

**Purpose**

To confirm the success of PCR by performing agarose
gel electrophoresis and visualizing DNA bands. Target DNA fragments are then extracted and purified from the gel for
use in **seamless cloning**. The final recombinant plasmid is transformed into **competent *E.
coli*** cells to enable propagation for further applications.

**Procedure**

**1. Agarose Gel Preparation and
Electrophoresis**

* Mix **2 g agarose** with **100 mL of TAE buffer** and water (1:1 ratio).
* Microwave until agarose is fully dissolved.
* Cool the solution slightly and add **YeaRed nucleic acid dye**.
* Pour the gel into a mold with a comb and allow to solidify completely.
* Place the gel into the electrophoresis tank and cover with TAE buffer.
* Mix each DNA sample with loading buffer at a **1:5 ratio**.
* Load DNA samples and a DNA Marker into the wells.
* Run electrophoresis at **180 V for 20 minutes**.
* Visualize the DNA bands using a **gel documentation system**.
* Identify and record the size of DNA fragments by comparing to the Marker.

**2. DNA Gel Extraction and
Purification**

* Excise the desired DNA band from the gel using a clean blade.
* Melt the gel slice and transfer it into a filter column.
* Centrifuge at **8000 rpm for 60 seconds**, discard flowthrough.
* Repeat filtration and centrifugation to remove gel debris.
* Wash the filter column twice with **washing buffer**, centrifuging each time at 8000 rpm for 60 sec.
* Open the column lid and centrifuge once more to remove residual ethanol.
* Transfer the column to a clean Eppendorf tube.
* Add **elution buffer**, incubate briefly, and centrifuge to collect purified DNA.
* Use a Nanodrop or spectrophotometer to measure **DNA concentration**.

**3. Seamless Cloning Reaction**

* In a tube, mix:
* Target fragment 1 (at 0.04 × its length, e.g., 36 ng)
* Target fragment 2 (at 0.02 × its length, e.g., 180 ng)
* 5× recombination buffer
* Homologous recombinase (enzyme:DNA ratio = 1:10)
* Incubate the mixture at **37°C for 30 minutes** to allow ligation into a circular plasmid.

**4. Transformation into Competent
Cells**

* Thaw competent *E. coli* cells on ice.
* Add recombinant plasmid mixture to cells and incubate on ice for **30 minutes**.
* Heat shocks the mixture at **42°C for 45 seconds**.
* Immediately chill on ice for **2 minutes**.
* Add **LB medium** and incubate at **37°C, 220 rpm for 1 hour** to allow recovery.
* Plate the bacteria on LB agar plates.
* Incubate plates overnight at **37°C** for colony growth.
* Perform **screening** to identify positive transformants.

![3](https://static.igem.wiki/teams/5734/notebook/3.webp)
![6ddc6a2e77b7eb324af89fe10e80e017](https://static.igem.wiki/teams/5734/notebook/4.webp)
![PD-L1原始图](https://static.igem.wiki/teams/5734/notebook/5.webp)

**Part 3: Colony PCR, Gel Electrophoresis, and
Strain Preservation**

**Purpose**

To confirm successful bacterial transformation by
performing PCR on selected colonies. Then, to visualize DNA bands through gel electrophoresis and preserve validated
bacterial strains for future use.

**Procedure**

**1. Colony Selection and Preservation**

* Pick individual bacterial colonies from transformation plates.
* Streak colonies onto new agar plates for backup.
* Inoculate selected colonies into LB broth with corresponding antibiotic.
* Shake at 37°C, 220 rpm to allow bacterial expansion.

**2. PCR Setup**

* In a microtube, prepare the PCR reaction:
  + Bacterial colony (as DNA template)
  + PCR buffer
  + Forward and reverse primers
  + dNTPs
  + DNA polymerase
  + Sterile distilled water
* Briefly centrifuge to collect the solution at the bottom.

**3. PCR Amplification**

* Denature DNA at **95°C** to separate strands.
* Anneal primers at approximately **50°C** to bind the template.
* Extend DNA strands at **72°C** using Taq polymerase and dNTPs.
* Store PCR products at 4°C or –20°C until analysis.

**4. Agarose Gel Electrophoresis**

* Prepare **1% agarose gel** using TAE buffer (0.5× or 1×).
* Heat the agarose in a microwave until fully dissolved.
* Cool to ~60°C, add DNA stain (1:10,000), and mix well.
* Pour into gel tray with comb and allow to solidify.
* Mix PCR products with loading dye (1:5 ratio).
* Load DNA samples and DNA Marker into the gel wells.
* Submerge gel in TAE buffer until just covered.
* Connect the electrodes (sample wells near the negative pole).
* Run electrophoresis at appropriate voltage (e.g., 100–180 V).
* Visualize bands under UV using a gel imager.

**5. Data Analysis and Strain Backup**

* Analyze the gel image to determine PCR success based on band size.
* Identify and mark successful transformants.
* Preserve confirmed positive colonies by streaking or freezing for long-term use.

**Part 4 Verification**

**Purpose:**
To verify successful
ligation and expression by plasmid extraction and electrophoresis, and to continue culturing positive bacterial
strains for sequencing validation.

**Procedures:**

* Inoculate single colonies into LB liquid medium with antibiotics in 12 mL culture tubes.
* Shake at 37°C, 220 rpm overnight.
* Transfer 1.5 mL of each culture to centrifuge tubes, spin down at 10,000 × g for 1 min.
* Perform plasmid extraction using commercial kit:
  + Resuspend in Solution A
  + Lyse with Solution B
  + Neutralize with Solution C
  + Bind DNA to column
  + Wash twice with wash buffer
  + Elute with TE buffer
* Measure plasmid concentration with Nanodrop.
* Prepare 1% agarose gel with TAE buffer and nucleic acid dye.
* Load plasmid DNA with loading dye and run electrophoresis with DNA Marker.
* Visualize bands under gel imager to confirm presence and size of plasmid.
* Store extracted plasmids at –20°C for sequencing.
* Preserve successful bacterial cultures by streaking on new plates or freezing glycerol stocks.

**Part 5 SDS-PAGE**

**Purpose:**
To extract and purify a
specific target protein (e.g., PD-1) from bacterial lysate.

**Procedures:**

**1. Protein Extraction**

* Add lysozyme and lysis buffer to bacterial pellet. Mix until fully resuspended.
* Place tube in an insulated container with ice. Sonicate at 50% power (200–300 W) with 5-second intervals, total of
  6 minutes.
* Centrifuge lysate at 1000 × g for 10 seconds at 4°C. Collect supernatant (crude protein extract).
* Prepare column in parallel by resuspending nickel affinity gel and equilibrating it with lysis buffer at 4°C.

**2. Protein Purification**

* Mix crude extract with equilibrated resin. Shake at 4°C for 1.5 hours.
* Load mixture into gravity column. Let drain slowly and repeat 3 times. Backup samples.
* Wash with washing buffer and collect flow-through (wash fractions).
* Elute protein with elution buffer. Collect elution fractions and label clearly.

**3. SDS-PAGE Validation**

* Assemble gel plates. Pour separating gel, level with ethanol, let set. Pour stacking gel and insert comb.
* Assemble gel into electrophoresis apparatus. Fill with Tris-Glycine-SDS buffer.
* Load samples: elution fractions, wash fractions, crude lysate, and protein ladder.
* Run at 80 V for 25 minutes, then 180 V for 35 minutes (~1 hour total).
* Stain gel and shake for 50 minutes. Image to confirm target protein (e.g., PD-1).

![28](https://static.igem.wiki/teams/5734/notebook/6.webp)
![32](https://static.igem.wiki/teams/5734/notebook/7.webp)

**Part 6 Transfer**

**Purpose:**
To transfer and detect
specific proteins using SDS-PAGE and PVDF membrane blotting.

**Procedures:**

**1: SDS-PAGE**

* Prepare and pour separating gel, level with ethanol, let polymerize. Pour stacking gel and insert comb.
* Mix Tris-Glycine-SDS buffer. Fill electrophoresis tank.
* Mix protein samples with loading buffer. Load samples and ladder.
* Run at 80 V for 25 min, then 180 V for 35 min (total ~1 hour).

**2: Protein Transfer**

* Cut off stacking gel. Soak PVDF in methanol for 5 minutes.
* Soak all blotting materials in transfer buffer.
* Assemble sandwich: sponge → filter paper → gel → PVDF → filter paper → sponge.
* Place sandwich in transfer tank with buffer and ice packs.
* Transfer at 150 mA constant current for 2 hours.

**Part 7 WB**

**Purpose:**
To validate the
expression of His-tagged proteins (e.g., PD-1/PD-L1) using SDS-PAGE and Western blot with HRP detection.

**Procedures:**

**1. SDS-PAGE**

* Lyse cells using lysis buffer and ultrasonic disruption. Centrifuge and collect protein.
* Mix with SDS loading buffer, heat to denature.
* Load into SDS-PAGE gel with marker.
* Fill tank with running buffer. Run 80 V for 25 min, then 180 V for 35 min.
* Prepare gel for transfer.

**2. Western Blot Transfer**

* Cut off stacking gel, soak PVDF in methanol.
* Soak all layers in transfer buffer.
* Assemble sandwich: sponge → filter paper → gel → membrane → filter paper → sponge.
* Place in transfer tank with buffer and ice. Run at 150 mA for 2 hours.

**3. Blocking and Detection**

* Block with 5% BSA for 1 hour at room temp.
* Wash with PBST 3–5 times.
* Incubate with Anti-His-HRP (1:3000) for 2 hours.
* Wash with PBST 5 times.
* Add TMB substrate. Observe color.
* Image using gel imager.

**Part 8 ELISA: Antibody Titer
Validation**

**Purpose:**
To verify the titer and
binding activity of antibodies specific to PD-1/PD-L1.

**Procedure:**

1. Coat wells with PD-1/PD-L1 proteins. Incubate at 4°C overnight.
2. Wash 3x with PBST. Block with 3% BSA for 1 hour. Wash 2x.
3. Add Anti-His HRP antibody, incubate 2 hours. Wash 3x.
4. Add TMB, incubate 20 minutes in dark. Add stop solution.
5. Measure absorbance at 650 nm with microplate reader.

![图片1](https://static.igem.wiki/teams/5734/notebook/8.webp)![图片3](https://static.igem.wiki/teams/5734/notebook/9.webp)

'
}]%}
{% block title %}Notebook{% endblock %}
{% block lead %}

NOTEBOOK

{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Overview",
"content": '

Cancer has emerged as a global public health
challenge. Current production of therapeutic antibodies often suffers from high costs, complex processes, and
limitations in expression levels and stability.Consequently, developing a safe, controllable, and efficient antibody
production system is crucial for advancing tumor therapeutics[1-3]. The red light-induced gene expression system, an
emerging regulatory technology, has demonstrated unique advantages in non-toxic regulation and spatiotemporally
precise expression. This study aims to develop an efficient and controllable nanobody production platform utilizing a
red light-induced gene expression system in Escherichia coli. By integrating red light-mediated gene regulation
technology with the robust protein production capacity of *E. coli*, we will achieve precise control over
nanobody expression for the preparation of anti-tumor drugs[4-6].

**Our design consists of two main
cycles:**

**Cycle 1: Establishment and Validation of Red
Light-Induced System**

Part 1: Construction of red light-induced system
(pRED vector)

Part 2: Development of red light-regulated GFP
expression system (pRED-GFP)

**Cycle 2: Nanobody Production Using the Red
Light System**

Part 1: Production of PD-L1 nanobody

Part 2: Production of PD-1 nanobody

In summary, Cycle 1 was designed to establish and
validate both the feasibility and sensitivity of the red light-induced expression system. Cycle 2 aimed to
systematically evaluate the nanobody production parameters under this optogenetic control, including yield
optimization, ideal production duration, light intensity requirements, and dissolved oxygen levels. These studies
provide fundamental theoretical support for our product-nanobody production kit.

'
},{
"title": "Cycle 1: Establishment and Validation of Red
Light-Induced System",
"content": '

**Part 1****Construction of red light-induced system pRED**

**Design:**

a red light-induced system, whose photosensory module
(PSM) has the bacterial phytochrome (DrBphP) as the key gene. The primary mechanisms are: a. Perception of red and
far-red light through the biliverdin chromophore. Biliverdin (BV) can be produced by heme oxygenase (HO) alone. b.
Light-induced structural changes in PSM are transmitted to the effector HK domain (YF1), thereby altering its basal
kinase and phosphatase activities. FixJ: an inverter cassette encoding the λ phage repressor cI and λ promoter pR. The
pRED plasmid was provided by our institution\'s strain repository, and the target gene *HO, DrBphP, YF1, FixJ*
were synthesized by a biotech company. The fragment was homologously recombined with a linearized plasmid to construct
the pRED(Figure 1).

![图片12](https://static.igem.wiki/teams/5734/engineering/1.webp)

Figure 1. The plasmid map of pRED

**Build:**

We obtained fragments for constructing plasmid pRED
using PCR technology, as shown in Figure 2a. We obtained fragments of approximately 4600bp and 4300bp in length, which
are consistent with our expected sizes. Subsequently, we seamlessly ligated the obtained target fragments and
transformed them into *Escherichia coli* DH5α, as shown in Figure 2b.

Furthermore, we randomly selected around 5 single
colonies from the obtained agar plates, sequenced the plasmids, and obtained the sequencing results of the correct
plasmid as shown in Figure 2d. Subsequently, after cultivation, we extracted the correct plasmid and transformed it
into *Escherichia coli* BL21, as shown in Figure 2c. Through this complete process, we successfully obtained
bacterial strains containing the correct plasmid pRED.

![图片16](https://static.igem.wiki/teams/5734/engineering/2.webp)

Figure 2. Construction results of plasmid pRED. a: PCR results; b: Transformation into DH5α after ligation; c:
Successful transformation of correct plasmid into BL21. d.Sequencing results of selected single colonies

**Part 2 Development of red light-regulated GFP
expression system pRED-GFP**

**Design:**

The construction of the pRED-GFP plasmid is based on
the plasmid pRED. Specific primers were designed to linearize the pRED plasmid. The GFP gene was provided by our
institution\'s strain repository. Through homologous recombination, the two fragments were ligated to form a plasmid,
resulting in the construction of pRED-GFP(Figure 2).

![图片1](https://static.igem.wiki/teams/5734/engineering/3.webp)

Figure 3. The plasmid map of pRED-GFP

**Build:**

PCR technology was employed to generate fragments for
the construction of plasmid pRED-GFP, with details presented in Figure 4a. A fragment of roughly 714 bp in length was
obtained, matching our anticipated size. Following this, the target fragments acquired were subjected to seamless
ligation, and the resulting product was transformed into *Escherichia coli* DH5α, as illustrated in Figure 4c.​

In addition, about 5 single colonies were randomly
picked from the obtained agar plates, and their plasmids were sequenced. The sequencing results of the correct plasmid
are displayed in Figure 4d. After cultivation, the correct plasmid was extracted and transformed into *Escherichia
coli* BL21, as shown in Figure 4b. Through this entire procedure, we successfully obtained bacterial strains
harboring the correct plasmid pRED.

.

![图片5](https://static.igem.wiki/teams/5734/engineering/4.webp)

Figure 4. Construction results of plasmid pRED-RED. a: PCR results; b: Successful transformation of correct plasmid
into BL21; c: Transformation into DH5α after ligation; d:
Sequencing results of selected single colonies

**Test**

To validate whether our constructed strains undergo
normal transcription and translation under red light induction, we conducted fluorescence intensity tests on the
strains under red light and dark conditions. We utilized strains containing the GFP gene, and the experimental results
demonstrate favorable outcomes in our experimental group (Figure 5). Only under red light induction did the strains
exhibit significantly high fluorescence intensity, confirming the feasibility of our strain modifications.

![](https://static.igem.wiki/teams/5734/engineering/5.webp)

Figure 5. Fluorescence intensity of the four strains under dark and red light conditions.

'
}, {
"title": "Cycle 2: Nanobody Production Using the Red
Light System",
"content": '

**Part 1: Production of PD-L1 nanobody
(pRED-PD-1 )**

**Design:**

The construction of the pRED-PD-1 plasmid is based on
the plasmid pRED. Specific primers were designed to linearize the pRED plasmid. The Anti-PD-1 was synthesized by a
biotech company. Through homologous recombination, the two fragments were ligated to form a plasmid, resulting in the
construction of pRED-PD-1(Figure 6).

![图片32](https://static.igem.wiki/teams/5734/engineering/6.webp)

Figure 6. The plasmid map of pRED-PD-1

**Build:**

We successfully amplified fragments for constructing
plasmid pRED-PD-1 via PCR technology, as depicted in Figure 7a. The resulting fragment, approximately 372 bp in size,
matched our expected length. Subsequent seamless ligation of the target fragments was performed, followed by
transformation into *Escherichia coli* DH5α, illustrated in Figure 7c.

Following this, we randomly picked approximately 5
individual colonies from the agar plates, sequenced the plasmids, and obtained sequencing data confirming the correct
plasmid, as displayed in Figure 7d. After cultivation, we isolated the correct plasmid and introduced it into
*Escherichia coli* BL21, as shown in Figure 7b. This comprehensive process led to the successful acquisition of
bacterial strains harboring the correct plasmid pRED.

.

![图片17](https://static.igem.wiki/teams/5734/engineering/7.webp)

Figure 7. Construction results of plasmid pRED-PD-1. a: PCR results; b: Successful transformation of correct plasmid
into BL21; c: Transformation into DH5α after ligation Sequencing results of selected single colonies; d: Sequencing
results of selected single colonies

**Part 2: Production of PD-1 nanobody
(pRED-PD-L1)**

**Design:**

The pRED-Anti-PD-L1 consists of eight components,
including BBa\_25OTICA0(PD-L1), BBa\_25CKVE78(HO),BBa\_25W1NO5 （DrBphP）;BBa\_25D3H7BT(YF1);BBa\_25CG7CJU(FixJ);BBa\_259SXEM0(λ repressor)and BBa\_25PPOVCP(pRED-Vector).
We constructed the pRED red light-inducible system using homologous recombination methods. Subsequently, this vector pRED was utilized to insert the nanobody PD-L1,
enabling the production of nanobodies.

The construction of the pRED-PD-L1 plasmid is based
on the plasmid pRED. Specific primers were designed to linearize the pRED plasmid. The Anti-PD-L1 was synthesized by a
biotech company. Through homologous recombination, the two fragments were ligated to form a plasmid, resulting in the
construction of pRED-GFP-Anti-PD-L1(Figure 8).

![图片25](https://static.igem.wiki/teams/5734/engineering/8.webp)

Figure 8. The plasmid map of pRED-PD-L1

**Build:**

Using PCR technology, we successfully obtained
fragments for the construction of plasmid pRED-PD-L1, depicted in Figure 9a. The fragment obtained was approximately
453 bp in length, aligning with our expected size. Subsequent to seamless ligation of the target fragments, they were
transformed into *Escherichia coli* DH5α, as illustrated in Figure 9b.

Moreover, approximately 5 single colonies were
randomly selected from the agar plates, and the plasmids were sequenced, yielding sequencing results confirming the
accuracy of the plasmid, as presented in Figure 9c. Following cultivation, the correct plasmid was extracted and
introduced into *Escherichia coli* BL21, as shown in Figure 9d. This comprehensive process culminated in the
successful acquisition of bacterial strains carrying the correct plasmid pRED.

![图片18](https://static.igem.wiki/teams/5734/engineering/9.webp)

Figure 9. Construction results of plasmid pRED-PD-L1. a: PCR results; b: Transformation into DH5α after ligation; c:
Sequencing results of selected single colonies; d: Successful transformation of correct plasmid into BL21

**Functional
Test**

1. **Protein Expression and Validation**

**1.1 SDS-PAGE for Anti-PD-1 and Anti-PD-L1**

Two strains of Escherichia coli BL21 carrying the
correct plasmids with Anti-PD-1 and Anti-PD-L1 were separately subjected to SDS-PAGE to verify the protein expression
of the target genes. The target proteins Anti-PD-1 and Anti-PD-L1 have sizes of 14.6 kDa and 16.2 kDa, respectively.
Protein expression was induced with 12W and performed at 25°C for 20 hours. Based on the results shown in Figure 10,
we can clearly observe protein bands of the target genes Anti-PD-1 and Anti-PD-L1 in both crude and purified proteins,
confirming the accuracy of our results.

![](https://static.igem.wiki/teams/5734/engineering/10.webp)

Figure 10. The SDS-PAGE results for Anti-PD-1 and Anti-PD-L1

**1.2 WB for Anti-PD-1 and Anti-PD-L1**

To further validate the expression and correct
molecular weights of our target proteins Anti-PD-1 and Anti-PD-L1, we conducted Western blot experiments on these two
strains. The experimental results, as shown in Figure 11, clearly demonstrate the expression of our target proteins at
lengths of 14.6 kDa and 16.2 kDa, confirming the accuracy of our protein expression.

![](https://static.igem.wiki/teams/5734/engineering/11.webp)

Figure 11. The WB results for Anti-PD-1 and Anti-PD-L1

2. **ELISA-Based
   Quantification of Nanobody Yield**

**2.1 ELISA analysis of production of two
nanobodies under different lighting times**

The duration of red light induction is a critical
factor influencing the production of nanobodies by the strains. In order to determine the detection time for
subsequent experiments, we first conducted experiments on two strains under different durations of red light
induction. Before testing the production of nanobodies in the fermentation broth, we tested the absorbance of our antibody when it completely reacted with different concentrations of PD-1 antigen, and drew the standard curve. Through the standard curve, we can quantitatively calculate the antibody yield.
Based on this, we conducted the quantification of nanobodies in the
fermentation broth. We ensured the reliability of our experimental data by diluting the samples at different
multiples. Through the experimental results (Figure 12), we explored the duration of light exposure at which the
strain\'s antibody production was maximized and did not decompose. Based on this experiment, we determined that in
subsequent experiments, samples would only be taken at 24 hours to measure antibody production.

![](https://static.igem.wiki/teams/5734/engineering/12-v1.webp)

Figure 12. The results of nanobody production under different light exposure durations.

**2.2 ELISA analysis of production of two
nanobodies under different lighting intensities**

To investigate the impact of light intensity on
nanobody production in the strains, we selected three intensities 3W, 6W, and 12W based on data from literature
reports for simultaneous induction of the strains, aiming to assess the effect of different red light intensities on
nanobody production. From the experimental results (Figure 13), it can be observed that when the red light irradiation
intensity increased from 3W to 6W, the production of the two antibodies increased by 10.53% and 16.06%, respectively.
However, upon further increase to 12W, there was minimal production enhancement, and the production of PD-L1 even
decreased by 4.44%. Therefore, for subsequent steps, we will utilize a red light intensity of 6W as our irradiation
intensity.

![](https://static.igem.wiki/teams/5734/engineering/13.webp)

Figure 13. The results of nanobody production under different lighting intensities.

**2.3 ELISA analysis of production of two
nanobodies under different oxygen conditions**

Because Escherichia coli is a facultative anaerobe,
it means that it can produce under both aerobic and anaerobic conditions. However, it is not clear whether the
production of nanobodies requires sufficient oxygen supply. We conducted experiments using two types of Erlenmeyer
flasks, as shown in Figure 14: a regular Erlenmeyer flask and a baffled Erlenmeyer flask, with the latter providing
more oxygen. This was done to provide different oxygen levels to the strains and assess the production of nanobodies.
The experimental results, as shown in Figure 15, revealed that as the dissolved oxygen level in the shake flask
increased, the production of antibodies PD-1 and PD-L1 increased by 17.30% and 6.56%, respectively.

We hypothesize that with the increase in dissolved
oxygen, the metabolism of the bacterial strain becomes more active, leading to higher antibody production.

![](https://static.igem.wiki/teams/5734/engineering/14.webp)

Figure 14. Different Erlenmeyer flasks - regular Erlenmeyer flask on the left, baffled Erlenmeyer flask on the right.

![](https://static.igem.wiki/teams/5734/engineering/15.webp)

Figure 15. The results of nanobody production under different oxygen conditions.

Table 1 presents the nanobody production yields
achieved under the established conditions (baffled flasks, 6W illumination, 24h induction period), with ELISA
measurements showing 2.289 ng/L for anti-PD-1 and 1.9485 ng/L for anti-PD-L1 nanobodies.

Table 1 . Nanobody production yield of PD-L1 and PD-1

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | PD-1(ng) | SD | PD-L1(ng) | SD |
| yield | 2.289 | 0.008485281 | 1.9485 | 0.009545942 |

**Learn**

1. The proteins we expressed are intracellular. In the future, we may consider incorporating signal peptides and
   specific transport proteins to transport the target antibodies to the extracellular space.
2. Currently, we have only used ELISA to verify the yield and efficacy of the nanobodies. In future studies, we will
   employ additional validation methods such as surface plasmon resonance (SPR) and animal experiments to further
   characterize the nanobodies\'s performance.
3. While our current focus has been on PD-1/PD-L1 nanobodies, we aim to establish a generalized platform capable of
   producing various nanobody classes, thereby validating the system\'s versatility.

'
},{
"title": "References",
"content": '

[1] Huang Z, Li Z, Zhang X, Kang S, Dong R, Sun L, Fu
X, Vaisar D, Watanabe K, Gu L. Creating Red Light-Switchable Protein Dimerization Systems as Genetically Encoded
Actuators with High Specificity. ACS Synth Biol. 2020 Dec 18;9(12):3322-3333. doi: 10.1021/acssynbio.0c00397. Epub
2020 Nov 12. PMID: 33179507; PMCID: PMC7749050.

[2] Zhou P, Jia Y, Zhang T, Abudukeremu A, He X,
Zhang X, Liu C, Li W, Li Z, Sun L, Guang S, Zhou Z, Yuan Z, Lu X, Yu Y. Red Light-Activated Reversible Inhibition of
Protein Functions by Assembled Trap. ACS Synth Biol. 2025 May 16;14(5):1437-1450. doi: 10.1021/acssynbio.4c00585. Epub
2025 Apr 30. PMID: 40304578.

[3] Liu M, Li L, Jin D, Liu Y. Nanobody-A versatile
tool for cancer diagnosis and therapeutics. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2021 Jul;13(4):e1697. doi:
10.1002/wnan.1697. Epub 2021 Jan 20. PMID: 33470555.

[4] Koch-Nolte F. Nanobody-based heavy chain
antibodies and chimeric antibodies. Immunol Rev. 2024 Nov;328(1):466-472. doi: 10.1111/imr.13385. Epub 2024 Aug 30.
PMID: 39212236; PMCID: PMC11659929.

[5] Tang X, Deng A, Chen W, Zhao Y, Wang M, Li C.
[Advances in nanobody screening technology]. Sheng Wu Gong Cheng Xue Bao. 2024 Feb 25;40(2):350-366. Chinese. doi:
10.13345/j.cjb.230472. PMID: 38369826.

[6] Salvador JP, Vilaplana L, Marco MP. Nanobody:
outstanding features for diagnostic and therapeutic applications. Anal Bioanal Chem. 2019 Mar;411(9):1703-1713. doi:
10.1007/s00216-019-01633-4. Epub 2019 Feb 8. PMID: 30734854.

'
}]%}
{% block title %}Engineering{% endblock %}
{% block lead %}

ENGINEERING

{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Disposition of Culture",
"content": '

**Goal:** Create a nutritive environment
for bacterial cultivation and microorganism activation.

**Materials:**

* Yeast Extract
* Sodium chloride
* dd H2O
* Sterilized duran bottle, cap
* Lab Refrigerator
* Tryptone
* Autoclave
* Agar

**Procedures:**

1. Prepare LB Medium (for *E. coli* recombinant strain maintenance/cultivation)
2. Measure 20g premixed LB broth powder (Tryptone:Yeast extract:NaCl = 2:1:1)
3. Note: For solid medium, add 1.5% agar
4. Transfer powder to sterile Duran bottle
5. Add dd H₂O to final volume of 1000 mL
6. Secure cap and autoclave (≥20 min at 121°C)
7. Cool to room temperature

'
},{
"title": "The Construction of Plasmid",
"content": '

**![图片10](https://static.igem.wiki/teams/5734/experiments/1.webp)**

1. **Construction of** **pRED**

1. **Obtaining the red light-induced system empty plasmid**

**Goal:** To construct the red
light-induced expression empty plasmid

**Materials:**

1) 2xPrimeStarMix

2) template

3) double distilled water (ddH2O)

4) Primer-R

5) Primer-F

6) centrifuge tube

**Procedures:**

1) Combine 25µL of 2xPrimerStarMix, 1µL of
template,
1µL of primer-R, and 1µL of primer-F in a centrifuge tube. Adjust the volume to 50µL with ddH2O.
Centrifuge
the mixture briefly if bubbles are present.

2) Transfer the mixture to a PCR thermal cycler
and
denature the fragments at 95˚C for 3 min.

3) Run 32 cycles consisting of 30 seconds
denaturation at 95˚C, 30 seconds annealing at 55˚C, and 2 min extension at 72˚C.

4) Perform a final extension step at 72˚C for 1
min.

5) Store the fragments at 4˚C if not used
immediately.

2. **Agarose gel construction, deployment and recycle**

**Goal:** To produce a functional
and
well-shaped gel for electrophoresis

**Materials:**

1) Casting Tray

2) Well comb

3) Microwave

4) 1x TAE

5) Agarose M

6) 10000x Nucleic acid gel stain

7) Erlenmeyer flask

**Procedures:**

1. Dissolve 0.3g of agarose in a 30mL Erlenmeyer flask.
2. Add TAE (running buffer) to the flask.
3. Heat the mixture using a microwave.
4. Melt the mixture in 30-second intervals.
5. Check for the formation of bubbles to ensure thorough mixing.
6. Incorporate 3μl of nucleic acid gel stain (10000x).
7. Allow the mixture to cool for 5-10 mins.
8. Transfer the agarose mixture into the casting tray.
9. Position the appropriate well comb to create wells in the gel.
10. Allow the gel to solidify for approximately 30 mins.
11. Remove the comb and transfer the gel to the gel box.

3. **Agarose gel electrophoresis**

**Goal:** To verify the DNA bands’
lengths of the plasmid extracted and recycle later.

**Procedures:**

1. Take out 50µl from each sample and mix with 5µl of loading buffer (10x).
2. Prepare the horizontal electrophoresis system for gel placement.
3. Mix 50µl of the combined sample solution until uniform in color.
4. Load the marker in the first well followed by 50µl of each sample into the respective wells.
5. Run the procedure at 120V for 20 mins.
6. Remove the gel from the apparatus and proceed with the subsequent steps.

4. **DNA Gel Extraction**

**Goal:** To recover the target
fragment
in the agarose gel.

**Materials:**

1) Agarose gel with target fragment

2) 1.5 mL EP tube

3) Agarose Gel Electrophoresis Recovery Kit

**Procedures:**

1. Excise the gel slice containing the 5984 bp fragment, removing excess gel, and transfer it to a 1.5 mL EP
   tube.
2. Add 500 microliters of buffer B2 and incubate the tube in a 50°C water bath until the gel is completely
   dissolved.
3. Transfer the melted gel solution into a purification column placed in a collection tube and centrifuge at 8000
   g
   for 30 seconds.
4. Discard the collection tube, reinsert the purification column, add 500 microliters of ethanol-containing wash
   solution, and centrifuge at 9000 g for 30 seconds. Empty the collection tube.
5. Repeat the wash step.
6. Centrifuge at 9000 g for 1 min, then briefly open the tube cap to allow ethanol evaporation for one min.
7. Transfer the purification column to a new 1.5 mL EP tube. Add 20 microliters of elution buffer to the center
   of
   the purification column.
8. Close the lid and incubate the tube for 1 min, followed by centrifugation at 9000 g for 1 min.
9. Discard the purification column and store the DNA in the EP tube at 4°C.

5. **Homologous recombination to construct plasmid pRED**

Goal: Constructing the target plasmid through
homologous recombination.

Materials:

1) ClonExpress Mix (2x)

2) ddH2O

3) PCR products of target fragment

Procedures:

1) Quantify the concentration of the
gel-recovered
fragments.

2) According to the instructions, prepare the
system.

3) Incubate at 50 ℃ for 15 mins.

6. **Transferring the recombined fragment into DH5α**

**Goal:** Transform the constructed
plasmid into *Escherichia coli* for validation and amplification.

**Materials:**

1) Plasmid after ligation

2) *E. coli* in a
competent state

**Procedures:**

1. Combine the ligated samples with *E. coli* samples.
2. Chill on ice for 20 mins.
3. Heat shock at 42 ℃ for 45 seconds, then promptly transfer to ice for 2-3 mins.
4. Mix with 900μL LB Agar solution and incubate for 30 mins.
5. Centrifuge at 5000xg for 3 mins, remove and discard 900μL of supernatant.
6. Apply the final product to agar plates and culture for 12-16 hours at 37 degrees Celsius.

7. **Colony PCR Validation**

**Goal:** Verify the transformants
and
inoculate them to grow with the target gRNA.

**Materials:**

1) Taq Master Mix (2x)

2) ddH2O

3) Culture plates post-transformation

**Procedures:**

1. Set up the system (20 μL total volume: 10 μL ClonExpress Mix, 1 μL primer-F, 1 μL primer-R, 8 μL ddH2O).
2. Using a sterile 2.5 μL pipette tip, pick a single clone and transfer it into the system.
3. Place the mixture into a PCR thermal cycler, following the PCR program according to the manufacturer\'s
   instructions.
4. Verify the successful construction of plasmid by performing agarose gel electrophoresis, where the presence of
   the
   correct bands confirms the outcome.

8. **Extracting the plasmid**

**Goal:** To obtain the plasmid pRED
from DH5α

**Materials:**

Plasmid Extraction Kit

**Procedures:**

1. In a 2 mL centrifuge tube, collect 2 mL of bacterial culture, centrifuge at 12000 rpm for 1 min, and remove
   the supernatant carefully.
2. Repeat the centrifugation step with another 2 mL of bacterial culture, discarding the supernatant afterward.
3. Resuspend the bacterial cell pellet in the tube by adding 250 μL of Solution I and vortexing thoroughly.
4. Introduce 250 μL of Solution II to the tube and gently invert it 6-8 times to lyse the bacteria completely.
5. Mix 350 μL of Solution III with the bacterial lysate, centrifuge at 12000 rpm for 10 min, and transfer the
   supernatant to a clean centrifuge tube.
6. Combine the supernatant from the previous step with 0.35 times the volume of anhydrous ethanol and mix well.
7. Apply the supernatant-ethanol mixture to an adsorbent column, allowing it to stand for 2 min, then centrifuge
   at 12000 rpm for 1 min, discarding the waste liquid.
8. Wash the column by adding 750 μL of rinse solution, centrifuging at 12000 rpm for 1 min, and discarding the
   waste solution.
9. Repeat the wash step with 700 μL of rinse solution, followed by centrifugation and discarding the waste
   solution.
10. Centrifuge the column at 12000 rpm for 2 min and let it sit at room temperature briefly.
11. Place the column in a clean tube, add 40 drops of eluent to the center of the adsorbent membrane, let it stand
    for 2 min at room temperature, and centrifuge at 12000 rpm for 1 min.

2. **Construction of** **pRED-GFP**

**A. Construction of pRED-GFP**

**Goal:** Obtain the expression
plasmid
carrying GFP.

**Materials:** Designed primers
specific
to pRED-GFP construction, keeping other materials consistent with the previous procedure.

**Procedures:** The GFP fragment and
the
vector were obtained through PCR. Subsequently, a plasmid was constructed using the homologous group method.
Followed
by transformation into competent *E. coli* DH5α.

**B. Transferring the recombined fragment
into
DH5α**

**Goal**: Transform the constructed
plasmid into *E. coli* for validation and amplification.

**Materials**: Consistent with the
above.

**Procedures:** Consistent with the
above.

**C. Colony PCR Validation**

**Goal:** Verify the transformants.

**Materials:** Consistent with the
above.

**Procedures:** Consistent with the
above.

3. **Construction of pRED-Anti-PD-1**

**A. Construction of
pRED-Anti-PD-1**

**Goal:** Construct a functional
plasmid
containing Anti-PD-1 on the expression plasmid.

**Materials:** Designed primers
specific
to pRED-Anti-PD-1 construction, keeping other materials consistent with the previous procedure.

**Procedures:** Transform the
ligated
plasmid into *E. coli* competent cells.

**B. Transferring the recombined fragment
into
DH5α**

**Goal:** Transform the constructed
plasmid into *E. coli* for validation and amplification.

**Materials:** Consistent with the
above.

**Procedures:** Consistent with the
above.

**C. Colony PCR Validation**

**Goal:** Verify the transformants.

**Materials:** Consistent with the
above.

**Procedures:** Consistent with the
above.

4. **Construction of pRED-Anti-PD-L1**

**A. Construction of
pRED-Anti-PD-L1**

**Goal:** Designed primers specific
to
pRED-Anti-PD-L1 construction, keeping other materials consistent with the previous procedure.

**Materials:** Designed primers
specific
to pRED-Anti-PD-L1 construction, keeping other materials consistent with the previous procedure.

**Procedures:** Transform the
ligated
plasmid into *E. coli* competent cells.

**B. Transferring the recombined fragment
into
DH5α**

**Goal:** Transform the constructed
plasmid into *E. coli* for validation and amplification.

**Materials:** Consistent with the
above.

**Procedures:** Consistent with the
above.

**C. Colony PCR Validation**

**Goal:** Verify the transformants.

**Materials:** Consistent with the
above.

**Procedures:** Consistent with the
above.

'
}, {
"title": "Functional Test",
"content": '

**![图片12](https://static.igem.wiki/teams/5734/experiments/2.webp)**

1. **The growth curve test of bacterial strains.**

**Goal:** Assessing the growth status
among different bacterial strains.

**Materials:**

1) ultraviolet spectrophotometer

2) pipette

**Procedures:**

1. Prepare the growth medium LB according to the specified recipe and sterilize it.
2. Inoculate the medium with the microbial to initiate growth (initial OD600 = 0.5).
3. Incubate the culture at the 37 ℃ with continuous monitoring.
4. At regular intervals, remove samples to measure the OD at a specific wavelength using a spectrophotometer.
5. Record the OD values to plot a growth curve showing the growth dynamics over time.
6. Analyze the growth curve to determine the growth phases and growth rate of the microbial culture.
7. Repeat the experiment with appropriate controls and replicates for accurate and reliable results.
8. Interpret the growth curve data to draw conclusions about the growth behavior of the microbial species under the
   experimental conditions.

2. **Fluorescence testing of GFP**

**Goal:** To validate the plasmid\'s
expression, GFP was cloned into the plasmid, followed by testing its expression in the bacterial strain.

**Materials:**

1) microplate reader

2) pipette

**Procedures:**

1. Inoculate 12 bacterial strains (4 plasmids with 3 replicates each, starting OD=0.5) and culture at 37°C,
   incubating under both dark and light-induction conditions.
2. Measure GFP expression intensity at 0, 2, 4, 6, 8, and 24 hours of cultivation (GFP excitation wavelength 488nm;
   emission wavelength 507nm).

3. **SDS-PAGE for Anti-PD-1 and Anti-PD-L1**

**Goal:** Evaluate the protein
expression levels of Anti-PD-1 and Anti-PD-L1.

**Materials:**

1) bacteria culture containing desired protein

2) 50 mL centrifuge tubes

3) beaker containing ice

4) centrifuge

5) sonicator

6) Sangon 12.5% SDS-PAGE Color Preparation kit

7) pipette

8) distilled water

9) electrophoresis buffer

10) protein ladder

11) vertical electrophoresis system

12) Coomassie blue

**Procedures:**

1) Transfer samples into 50 mL centrifuge tubes and
centrifuge at 4000 rpm for 20 mins. Place a centrifuge tube partially submerged in an ice-filled beaker, then place
the beaker and tube into the sonicator.

2) Sonicate for 15 mins. Repeat the sonication
process for all samples. Centrifuge samples at 4000 rpm for 15 mins. Discard the cell fragments collected at the
bottom of the tubes. The remaining supernatant will contain the desired proteins.

3) Combine 2.2 mL of 2X separating gel solution,
2.2
mL of 2X separating gel buffer, and 44 μL of catalyst in a plastic cup using a pipette.

4) Carefully inject the mixture into the casting
stand and frame to prevent bubble formation.

5) Add 1 mL of water to level the top surface.
Allow
the gel to set for 8 mins. Remove the water layer from the top of the gel.

6) Mix 825 μL of 2X stacking gel solution, 825 μL
of
2X stacking gel buffer, and 11 μL of catalyst in a plastic cup using a pipette.

7) Fill the cast entirely with the stacking gel
mixture, gently insert the comb without introducing air bubbles.

8) Let the gel set for 12 mins, remove the comb
carefully, and rinse the wells with electrophoresis buffer.

9) Load the protein ladder into the first well,
followed by sample loading in subsequent wells.

10) Transfer the gel into a vertical
electrophoresis
system.

11) Set the voltage to 120V and run the gel for 90
mins. Stain the gel with Coomassie blue by immersing it for 30 mins.

4. **WB for Anti-PD-1 and Anti-PD-L1**

**Goal:** For the detection of target
proteins.

**Materials:**

1) WB kit

2) pipette

**Procedures:**

1) Cell lysis: Use lysis buffer to break the cells
and release proteins.

2) SDS-PAGE gel electrophoresis: Load protein
samples
onto SDS-PAGE gel to separate proteins based on molecular weight.

3) Transfer proteins from gel to membrane: Use a
wet
transfer method to transfer separated proteins to a PVDF membrane.

4) Blocking: Block nonspecific binding sites with a
protein blocking agent.

5) Primary antibody incubation: Incubate the
membrane
with a specific primary antibody to allow binding to the target protein.

6) Washing: Wash the membrane with TBST to remove
unbound antibody.

7) Secondary antibody incubation: Incubate the
membrane with an HRP-conjugated secondary antibody to form a complex with the primary antibody.

8) Washing: Wash the membrane again with TBST to
remove unbound secondary antibody.

9) Visualization and imaging: Use a staining method
to visualize protein bands, followed by imaging and analysis.

5. **ELISA analysis of production of two nanobodies under different lighting times**

**![图片13](https://static.igem.wiki/teams/5734/experiments/3.webp)**

**Goal:** Quantitative analysis of the
antibody production of two antibody-producing strains under different red light induction conditions.

**Materials:**

1) microplate reader

2) pipette

3) ELISA KIT

4) red light

**Procedures:**

1) Inoculate two bacterial strains separately into
conical flasks with an initial OD of 0.5, with three replicates for each strain. Subsequently, collect samples at
different time points and store the samples in a -20°C freezer.

2) rhTNF-α Coating: Coat microwells with 100 μL per
well (2μg/mL, 200ng in total) of PD-1/PD-L1diluted in Coating Buffer (1×PBS). Seal plate and incubate overnight at
4°
C.

3) Wash Plate: Aspirate wells and wash 3 times with
≥
300 μL/well Wash Buffer (1×PBS containing 0.05% Tween20). After last wash, invert plate and blot on absorbent paper
to
remove any residual buffer.

3) Blocking: Block plates with ≥ 200 μL/well
Blocking
Buffer. Incubate at RT for 1 hour.

4) Washing Plate: Repeat step (3).

5) Prepare tested sample dilutions in Assay Diluent
Buffer.

6) Sample Incubation: Pipette 100 μL of each
sample,
and control into wells. Seal plate and incubate for 2 hours at RT or 37° C 1hr.

7) Washing Plate: Aspirate/ wash as in step 2, but
with 5 total washes.

8) Detection Ab Incubation: Add 100μL of His Tag
Mouse Manodonal Antibody (HRP Conjugated)(in Assay Diluent Buffer) to each well. Seal plate and incubate for 1 hour
at
RT. Note: The Conc. Of anti-His Tag mAb-HRP should be optimized.

9) Washing Plate: Repeat step (8).

10) Detection: Add 100 μL of TMB Substrate Solution
to each well. Incubate plate (without plate sealer) for 10-30 minutes at room temperature in the dark. Note: The
best
color development time will fluctuated based on different exp. context. The longest color development time is not
more
than 30 minutes.

11) Stop Reaction: Add 100 μL of TMB Stop Solution
(650nm) to each well.

12) Data Collection: Read absorbance at 650 nm
within
30 minutes of stopping reaction.

6. **ELISA analysis of production of two nanobodies under different lighting intensities**

**Goal:** Quantitative analysis of the
antibody production of two antibody-producing strains under different lighting intensities conditions.

**Materials:**

Consistent with the above.

**Procedures:** Inducing bacterial
strains by irradiation with red light at different power levels, keeping other materials consistent with the
previous
procedure.

7. **ELISA analysis of production of two nanobodies different oxygen conditions**

**Goal:** Quantitative analysis of the
antibody production of two antibody-producing strains under different oxygen conditions.

**Materials:**

Consistent with the above.

**Procedures:** Fermentation in
conical
flasks under different oxygen conditions (with baffle and without baffle, keeping other materials consistent with
the
previous procedure.

'
}]%}
{% block title %}Experiments{% endblock %}
{% block lead %}

EXPERIMENTS

{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Overview",
"content": '

Lung cancer is one of the most common malignant
tumors worldwide. Among these, non-small cell lung cancer accounts for approximately 80% of all lung cancer cases.
Current main methods for treating cancer: antibody drugs, among which nanobodies have garnered significant attention
in anti-tumor therapy due to their high affinity, low immunogenicity, and good tissue penetration[1]. This study
selects non-small cell lung cancer as the therapeutic target, focusing on developing nanobodies against PD-L1 and
PD-1[2-5]. We leverage a red light-inducible gene expression system in Escherichia coli to develop a high-yield,
tunable nanobody biomanufacturing pipeline.

First, construct a red light-induced system, whose
photosensory module (PSM) has the bacterial phytochrome (DrBphP) as the key gene. The primary mechanisms are(Figure
1)[6]:

1. Perception of red and far-red light through the biliverdin chromophore. Biliverdin (BV) can be produced by heme
   oxygenase (HO) alone.
2. Light-induced structural changes in PSM are transmitted to the effector HK domain (YF1), thereby altering its
   basal kinase and phosphatase activities.
3. FixJ: an inverter cassette encoding the λ phage repressor cI and λ promoter pR. We integrated a GFP reporter
   module into the genetic cassette to validate the sensitivity of the red light-inducible system.

![图片15](https://static.igem.wiki/teams/5734/results/1.webp)

Figure 1.The composition and principle of the red light induction system

The second is nanobody production and verification:
clone Anti-PD-L1 and Anti-PD-1 nanobody genes into expression vectors, transform BL21 *E. coli* strains
respectively. After red light-induced expression, employ dual verification with ELISA and functional experiments:
confirm the red light system can effectively drive antibody production. The third is production condition
optimization: systematically investigate the effects of parameters such as light intensity and duration on antibody
yield, establishing relatively ideal production conditions.

'
},{
"title": "Contents",
"content": '

1. Construction of plasmids

1.1 Construction of pRED

1.2 Construction of pRED-GFP

1.3 Construction of pRED-Anti-PD-1

1.4 Construction of pRED-Anti-PD-L1

2. Functional Test

2.1 Red light system sensitivity verification

2.1.1The growth curve test of bacterial strains

2.1.2 Fluorescence testing of GFP

2.2 Protein expression and verification

2.2.1 SDS-PAGE for Anti-PD-1 and Anti-PD-L1

2.2.2WB for Anti-PD-1 and Anti-PD-L1

2.3 ELISA Validation of nanobody yield and production
conditions

2.3.1 ELISA analysis of production of two nanobodies
under different lighting times

2.3.2ELISA analysis of production of two nanobodies
under different lighting intensities

2.3.3ELISA analysis of production of two nanobodies
under different oxygen conditions

3. Future plans
4. References

',
"children": [{
"title": "1. Construction of plasmids",
"content": '

**1.1 Construction of
pRED**

We obtained fragments for constructing plasmid pRED
using PCR technology, as shown in Figure 2a. We obtained fragments of approximately 4600bp and 4300bp in length, which
are consistent with our expected sizes. Subsequently, we seamlessly ligated the obtained target fragments and
transformed them into *Escherichia coli* DH5α, as shown in Figure 2b.

Furthermore, we randomly selected around 5 single
colonies from the obtained agar plates, sequenced the plasmids, and obtained the sequencing results of the correct
plasmid as shown in Figure 2d. Subsequently, after cultivation, we extracted the correct plasmid and transformed it
into *Escherichia coli* BL21, as shown in Figure 2c. Through this complete process, we successfully obtained
bacterial strains containing the correct plasmid pRED.

![图片16](https://static.igem.wiki/teams/5734/results/2.webp)

Figure 2. Construction results of plasmid pRED. a: PCR results; b: Transformation into DH5α after ligation; c:
Successful transformation of correct plasmid into BL21. d.Sequencing results of selected single colonies

**1.2 Construction of pRED-GFP**

PCR technology was employed to generate fragments for
the construction of plasmid pRED-GFP, with details presented in Figure 3a. A fragment of roughly 714 bp in length was
obtained, matching our anticipated size. Following this, the target fragments acquired were subjected to seamless
ligation, and the resulting product was transformed into *Escherichia coli* DH5α, as illustrated in Figure 3c.​

In addition, about 5 single colonies were randomly
picked from the obtained agar plates, and their plasmids were sequenced. The sequencing results of the correct plasmid
are displayed in Figure 3d. After cultivation, the correct plasmid was extracted and transformed into *Escherichia
coli* BL21, as shown in Figure 3b. Through this entire procedure, we successfully obtained bacterial strains
harboring the correct plasmid pRED.

.

![图片5](https://static.igem.wiki/teams/5734/results/3.webp)

Figure 3. Construction results of plasmid pRED-GFP. a: PCR results; b: Successful transformation of correct plasmid
into BL21

c: Transformation into DH5α after ligation; d:
Sequencing results of selected single colonies

**1.3 Construction of pRED-PD-1**

We successfully amplified fragments for constructing
plasmid pRED-PD-1 via PCR technology, as depicted in Figure 4a. The resulting fragment, approximately 372 bp in size,
matched our expected length. Subsequent seamless ligation of the target fragments was performed, followed by
transformation into *Escherichia coli* DH5α, illustrated in Figure 4c.

Following this, we randomly picked approximately 5
individual colonies from the agar plates, sequenced the plasmids, and obtained sequencing data confirming the correct
plasmid, as displayed in Figure 4d. After cultivation, we isolated the correct plasmid and introduced it into
*Escherichia coli* BL21, as shown in Figure 4b. This comprehensive process led to the successful acquisition of
bacterial strains harboring the correct plasmid pRED.

.

![图片17](https://static.igem.wiki/teams/5734/results/4.webp)

Figure 4. Construction results of plasmid pRED-PD-1. a: PCR results; b: Successful transformation of correct plasmid
into BL21; c: Transformation into DH5α after ligation Sequencing results of selected single colonies; d: Sequencing
results of selected single colonies

**1.4 Construction of pRED-PD-L1**

Using PCR technology, we successfully obtained
fragments for the construction of plasmid pRED-PD-L1, depicted in Figure 5a. The fragment obtained was approximately
453 bp in length, aligning with our expected size. Subsequent to seamless ligation of the target fragments, they were
transformed into *Escherichia coli* DH5α, as illustrated in Figure 5b.

Moreover, approximately 5 single colonies were
randomly selected from the agar plates, and the plasmids were sequenced, yielding sequencing results confirming the
accuracy of the plasmid, as presented in Figure 5c. Following cultivation, the correct plasmid was extracted and
introduced into *Escherichia coli* BL21, as shown in Figure 5d. This comprehensive process culminated in the
successful acquisition of bacterial strains carrying the correct plasmid pRED.

![图片18](https://static.igem.wiki/teams/5734/results/5.webp)

Figure 5. Construction results of plasmid pRED-PD-L1. a: PCR results; b: Transformation into DH5α after ligation; c:
Sequencing results of selected single colonies; d: Successful transformation of correct plasmid into BL21

'
},{
"title": "2. Functional Test",
"content": '

**2.1 Red light system sensitivity
verification**

2.1.1. The growth curve test of bacterial strains

The four constructed plasmids were individually
transformed into *E.coli* BL21 strains, and growth curves of these four strains were determined.
OD600 measurements were taken at 0, 2, 4, 6, 8, 12, and 24 hours under two different conditions: darkness
and red light induction, with three replicates for each condition. These experimental settings allowed us to assess
the impact of plasmid introduction and gene expression on strain growth, providing fundamental guidance for subsequent
experiments. From the experimental results shown in Figure 6, it is evident that under both light induction conditions
and strain expression, the growth of the four strains remained relatively consistent. This provides valuable insights
for our follow-up experiments.

![](https://static.igem.wiki/teams/5734/results/6.webp)

Figure 6. Growth curves of the four strains under dark and red light conditions.

2.1.2 Fluorescence testing of GFP

To validate whether our
constructed strains undergo normal transcription and translation under red light induction, we conducted fluorescence
intensity tests on the strains under red light and dark conditions. We utilized strains containing the GFP gene, and
the experimental results demonstrate favorable outcomes in our experimental group (Figure 7). Only under red light
induction did the strains exhibit significantly high fluorescence intensity, confirming the feasibility of our strain
modifications.

![](https://static.igem.wiki/teams/5734/results/7.webp)

Figure 7. Fluorescence intensity of the four strains under dark and red light conditions.

**2.2 Protein expression and
verification**

2.2.1 SDS-PAGE for Anti-PD-1 and Anti-PD-L1

Two strains of Escherichia coli BL21 carrying the
correct plasmids with Anti-PD-1 and Anti-PD-L1 were separately subjected to SDS-PAGE to verify the protein expression
of the target genes. The target proteins Anti-PD-1 and Anti-PD-L1 have sizes of 14.6 kDa and 16.2 kDa, respectively.
Protein expression was induced with12W red and performed at 25°C for 20 hours. Based on the results shown in Figure 8,
we can clearly observe protein bands of the target genes Anti-PD-1 and Anti-PD-L1 in both crude and purified proteins,
confirming the accuracy of our results.

![](https://static.igem.wiki/teams/5734/results/8.webp)

Figure 8. The SDS-PAGE results for Anti-PD-1 and Anti-PD-L1

2.2.2 WB for Anti-PD-1 and Anti-PD-L1

To further validate the expression and correct
molecular weights of our target proteins Anti-PD-1 and Anti-PD-L1, we conducted Western blot experiments on these two
strains. The experimental results, as shown in Figure 9, clearly demonstrate the expression of our target proteins at
lengths of 14.6 kDa and 16.2 kDa, confirming the accuracy of our protein expression.

![](https://static.igem.wiki/teams/5734/results/9.webp)

Figure 9. The WB results for Anti-PD-1 and Anti-PD-L1

**2.3 ELISA Validation of nanobody yield and
production conditions**

2.3.1 ELISA analysis of production of two nanobodies
under different lighting times

The duration of red light induction is a critical
factor influencing the production of nanobodies by the strains. In order to determine the detection time for
subsequent experiments, we first conducted experiments on two strains under different durations of red light
induction. Before testing the production of nanobodies in the fermentation broth, we tested the absorbance of our antibody when it completely reacted with different concentrations of PD-1/PD-L1 antigen, and drew the standard curve. Through the standard curve, we can quantitatively calculate the antibody yield.
Based on this, we conducted the quantification of nanobodies in the
fermentation broth. We ensured the reliability of our experimental data by diluting the samples at different
multiples. Through the experimental results (Figure 10), we explored the duration of light exposure at which the
strain\'s antibody production was maximized and did not decompose. Based on this experiment, we determined that in
subsequent experiments, samples would only be taken at 24 hours to measure antibody production.

![](https://static.igem.wiki/teams/5734/results/10-v1.webp)

Figure 10. The results of nanobody production under different light exposure durations.

2.3.2 ELISA analysis of production of two nanobodies
under different lighting intensities

To investigate the impact of light intensity on
nanobody production in the strains, we selected three intensities 3W, 6W, and 12W based on data from literature
reports for simultaneous induction of the strains, aiming to assess the effect of different red light intensities on
nanobody production. From the experimental results (Figure 11), it can be observed that when the red light irradiation
intensity increased from 3W to 6W, the production of the two antibodies increased by 10.53% and 16.06%, respectively.
However, upon further increase to 12W, there was minimal production enhancement, and the production of PD-L1 even
decreased by 4.44%. Therefore, for subsequent steps, we will utilize a red light intensity of 6W as our irradiation
intensity.

![](https://static.igem.wiki/teams/5734/results/11.webp)

Figure 11. The results of nanobody production under different lighting intensities.

2.3.3 ELISA analysis of production of two nanobodies
under different oxygen conditions

Because Escherichia coli is a facultative anaerobe,
it means that it can produce under both aerobic and anaerobic conditions. However, it is not clear whether the
production of nanobodies requires sufficient oxygen supply. We conducted experiments using two types of Erlenmeyer
flasks, as shown in Figure 12: a regular Erlenmeyer flask and a baffled Erlenmeyer flask, with the latter providing
more oxygen. This was done to provide different oxygen levels to the strains and assess the production of nanobodies.
The experimental results, as shown in Figure 13, revealed that as the dissolved oxygen level in the shake flask
increased, the production of antibodies PD-1 and PD-L1 increased by 17.30% and 6.56%, respectively.

We hypothesize that with the increase in dissolved
oxygen, the metabolism of the bacterial strain becomes more active, leading to higher antibody production.

![](https://static.igem.wiki/teams/5734/results/12.webp)

Figure 12. Different Erlenmeyer flasks - regular Erlenmeyer flask on the left, baffled Erlenmeyer flask on the right.

![](https://static.igem.wiki/teams/5734/results/13.webp)

Figure 13. The results of nanobody production under different oxygen conditions.

Table 1 presents the nanobody production yields
achieved under the established conditions (baffled flasks, 6W illumination, 24h induction period), with ELISA
measurements showing 2.289 ng/L for anti-PD-1 and 1.9485 ng/L for anti-PD-L1 nanobodies.

Table 1 . Nanobody production yield of PD-L1 and PD-1

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | PD-1(ng) | SD | PD-L1(ng) | SD |
| yield | 2.289 | 0.008485281 | 1.9485 | 0.009545942 |

'
},{
"title": "3. Future plans",
"content": '

In our study, we only expressed individual key
production genes in experiments. In future experiments, we can try genes from more sources. The proteins we expressed
are intracellular. In the future, we may consider incorporating signal peptides and specific transport proteins to
transport the target antibodies to the extracellular space. These two directions will be the focus of our future
research. To further optimize the production of nanobodies and provide theoretical support for this field, we will
conduct more comprehensive research on this topic in the future.

'
}]
}, {
"title": "References",
"content": '

[1] Liu Y, Huang H. Expression of single-domain
antibody in different systems. Appl Microbiol Biotechnol. 2018 Jan;102(2):539-551. doi: 10.1007/s00253-017-8644-3.
Epub 2017 Nov 25. PMID: 29177623.

[2] Meihui Cui, Tao Sun, Shubin Li, Huizhuo Pan, Jing
Liu, Xinyu Zhang, Lianyue Li, Shanshan Li, Chunyang 2. 2. Wei, Chengzhuang Yu, Chun Yang, Ning Ma, Binglin Ma,
Shenjunjie Lu, Jin Chang, Weiwen Zhang, Hanjie Wang,NIR light-responsive bacteria with live bio-glue coatings for
precise colonization in the gut,Cell Reports,

[3] Volume 36, Issue 11,2021,109690,ISSN 2211-1247,
<https://doi.org/10.1016/j.celrep.2021.109690.>

[4] Cai H, Yao H, Li T, et al. An improved
fluorescent tag and its nanobodies for membrane protein expression, stability assay, and purification. Commun Biol,
2020, 3(1): 753

[5] Li C, Tang Z O, Hu Z X, et al. Natural
single-domain antibody-nanobody: a novel concept in the antibody field. J Biomed Nanotechnol, 2018, 14(1): 1-19.

[6] Multamäki E, García de Fuentes A, Sieryi O, Bykov
A, Gerken U, Ranzani AT, Köhler J, Meglinski I, Möglich A, Takala H. Optogenetic Control of Bacterial Expression by
Red Light. ACS Synth Biol. 2022 Oct 21;11(10):3354-3367. doi: 10.1021/acssynbio.2c00259.

'}]%}
{% block title %}Results{% endblock %}
{% block lead %}

RESULTS

{% endblock %}