1. HO, BBa_25CKVE78

Name: HO

Base Pairs: 320 bp

Origin: D. radiodurans(strain DSMZ-20359)

Usage and Biology

Heme oxygenase is utilized in the breakdown of heme, a process that releases biliverdin, carbon monoxide, and iron. In research and biotechnology, heme oxygenase may be used to study heme metabolism, investigate the effects of heme breakdown products, or develop therapeutic strategies targeting heme-related disorders. Heme oxygenase is a critical enzyme involved in the catabolism of heme, an essential component of hemoglobin and other heme-containing proteins. The expression and regulation of heme oxygenase are tightly controlled in response to cellular heme levels, oxidative stress, and various signaling pathways in the body[1].

2. DrBphP, BBa_25W1NO5O

Name: DrBphP

Base Pairs: 1286 bp

Origin: Deinococcus radiodurans

Usage and Biology

DrBphP is utilized as a photoreceptor that can sense red/far-red light and trigger downstream signaling pathways in response to light stimuli. Bacterial phytochromes, such as DrBphP, contain light-absorbing chromophores that undergo conformational changes upon light absorption. DrBphP and related bacterial phytochromes play a role in regulating bacterial physiology in response to environmental light conditions, such as adjusting growth, metabolism, or biofilm formation[2].

3. YF1, BBa_25D3H7BT

Name: YF1

Base Pairs: 530 bp

Origin: D. radiodurans(strain DSMZ-20359)

Usage and Biology

The YF1 domain is utilized as a sensor domain in two-component signal transduction systems, where it detects specific signals and initiates a signaling cascade. Upon phosphorylation, the HK domain interacts with response regulators to relay signals and regulate gene expression, metabolism, or other cellular processes. YF1 and other effector Histidine Kinase domains play a critical role in cellular adaptation to changing conditions, including osmolarity, light, and nutrient availability[1-2].

4. FixJ, BBa_25CG7CJU

Name: FixJ

Base Pairs: 439 bp

Origin: B. japonicum

Usage and Biology

FixJ is a response regulator protein that plays a crucial role in certain bacteria, particularly in the process of nitrogen fixation. Typically functioning as a response regulator in two-component signal transduction systems, FixJ interacts with the effector component to modulate gene expression and cellular functionsy[1-2].

5. GFP, BBa_25IS05UV

Name: GFP

Base Pairs: 714 bp

Origin: previous iGEM part

Usage and Biology

GFP is commonly utilized as a fluorescent marker in biological research to visualize and track proteins, cells, and organelles in living systems. It serves as a valuable tool for studying gene expression, protein localization, cell dynamics, and molecular interactions. GFP functions as a reporter protein that emits green fluorescence when exposed to ultraviolet or blue light. The protein's structure includes a chromophore that undergoes autocatalytic reactions to produce the characteristic green fluorescence. GFP has been engineered into numerous variants with different spectral properties, allowing for multicolor labeling and advanced imaging techniques[3].

6. Anti-PD-1, BBa_25VZ0DMO

Name: Anti-PD-1

Base Pairs: 372 bp

Origin: Anti-PD-1 antibodies are typically engineered monoclonal antibodies developed in the laboratory through biotechnological methods.

Usage and Biology

Anti-PD-1 antibodies are used in cancer immunotherapy to help the immune system recognize and attack cancer cells(Figure 1). By blocking the PD-1 receptor, these antibodies prevent cancer cells from evading immune detection and enhance the immune response against tumors. The PD-1 pathway is a key immune checkpoint that regulates the immune response to prevent excessive activation and maintain self-tolerance. Cancer cells can exploit this pathway to suppress the immune system's ability to recognize and destroy them. Anti-PD-1 antibodies disrupt this immune checkpoint, allowing the immune system to better target and eliminate cancer cells[4-5].



Figure 1. The DNA sequence of PD-1

7. Anti-PD-L1, BBa_25OTICA0

Summary

Based on BBa_K5526008 (PD-L1), we constructed a new combination plasmid (pRED-PD-L1 ) by attaching to the carrier (pRED) which we created as a red control system. By combination of the PD-L1 protein expression and the red-based regulation engineering of pRED, we can form a red-based switch thereby preventing the leakage of engineered microorganisms, which expand the usage of ccdB and make a contribution to the biosafety career. This approach not only enhancing the yield of nanobodies, which improve nanobody PD-L1 purity.

Documentation:

a. Usage and Biology:

Anti-PD-L1 antibodies are used in cancer immunotherapy to block the interaction between PD-L1(Figure 2) on cancer cells and PD-1 on immune cells. By inhibiting this interaction, Anti-PD-L1 antibodies help prevent cancer cells from evading immune detection and enhance the immune response against tumors. The PD-L1 pathway is a crucial mechanism used by cancer cells to evade immune surveillance by inhibiting T cell activity. By blocking PD-L1, Anti-PD-L1 antibodies restore the ability of immune cells to recognize and attack cancer cells, leading to an enhanced anti-tumor immune response[5-7].



Figure 2. The DNA sequence of PD-L1

pRED carries the light-sensing module (PSM) containing the key gene bacteriophytochrome (DrBphP), which perceives red and far-red light through the biliverdin (BV) chromophore generated by heme oxygenase (HO). The light-induced structural changes in PSM are transmitted to the effector Histidine Kinase domain (YF1), altering its kinase and phosphatase activities. Additionally, pRED includes FixJ, encoding an inverted box of genes for the λ phage repressor protein cI and the λ promoter pR (Figure 3). We constructed a novel red light-induced nanobody production platform and utilized this system to produce PD-L1 nanobodies, achieving enhanced antibody yield.



Figure 3. Principle of Red Light-Induced PD-L1 Nanobody Production

Characterization/Measurement

1. Protein expression and verification

1.1 SDS-PAGE for Anti-PD-L1

Two strains of Escherichia coli BL21 carrying the correct plasmids with Anti-PD-L1 were separately subjected to SDS-PAGE to verify the protein expression of the target genes. The target proteins Anti-PD-L1 have sizes of 16.2 kDa, respectively. Protein expression was induced with 12W Red and performed at 25°C for 20 hours. Based on the results shown in Figure 4, we can clearly observe protein bands of the target genes Anti-PD-L1 in both crude and purified proteins, confirming the accuracy of our results.To further validate the expression and correct molecular weights of our target proteins Anti-PD-L1, we conducted Western blot experiments on these two strains. The experimental results, as shown in Figure 4, clearly demonstrate the expression of our target proteins at lengths of 16.2 kDa, confirming the accuracy of our protein expression.



Figure 4. The SDS-PAGE and WB results for Anti-PD-L1

2. ELISA Validation of nanobody yield and production conditions

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 (8h,24h and 30h) of red light induction at 25°C. 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-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 5), we explored the duration of light exposure at which the strain's antibody production was maximized and did not decompose. Figure 5 shows that PD-L1 nanobody production at both 24h and 30h was significantly higher than at 8h, but there was no significant difference between the 24h and 30h yields. Based on this experiment, we determined that in subsequent experiments, samples would only be taken at 24 hours to measure antibody production at 25°C .



Figure 5. 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 6), 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.



Figure 6. 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 7: 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 8, 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.



Figure 7. Different Erlenmeyer flasks - regular Erlenmeyer flask on the left, baffled Erlenmeyer flask on the right.



Figure 8. The results of nanobody production under different oxygen conditions.

Table 2 presents the nanobody production yields achieved under the established conditions (baffled flasks, 6W illumination, 24h induction period), with ELISA measurements showing 1.9485 ng/L for anti-PD-L1 nanobodies.

Table 2 . Nanobody production of PD-L1

	PD-L1(ng)	SD
yield	1.9485	0.009545942

1. pRED, BBa_25U18KUJ

Construction Design

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 9).



Figure 9. The plasmid map of pRED

Engineering Principle

pRED carries the light-sensing module (PSM) containing the key gene bacteriophytochrome (DrBphP), which perceives red and far-red light through the biliverdin (BV) chromophore generated by heme oxygenase (HO). The light-induced structural changes in PSM are transmitted to the effector Histidine Kinase domain (YF1), altering its kinase and phosphatase activities. Additionally, pRED includes FixJ, encoding an inverted box of genes for the λ phage repressor protein cI and the λ promoter pR (Figure 10).



Figure 10.Composition and Principle of the Red Light-Induced System

Experimental Approach

We obtained fragments for constructing plasmid pRED using PCR technology, as shown in Figure 11a. 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 11b.

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 11d. Subsequently, after cultivation, we extracted the correct plasmid and transformed it into Escherichia coli BL21, as shown in Figure 11c. Through this complete process, we successfully obtained bacterial strains containing the correct plasmid pRED.



Figure 11. 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

2. pRED-GFP, BBa_25SM97QW

Construction 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 12).



Figure 12. The plasmid map of pRED-GFP

Engineering Principle

pRED carries the light-sensing module (PSM) containing the key gene bacteriophytochrome (DrBphP), which perceives red and far-red light through the biliverdin (BV) chromophore generated by heme oxygenase (HO). The light-induced structural changes in PSM are transmitted to the effector Histidine Kinase domain (YF1), altering its kinase and phosphatase activities. Additionally, pRED includes FixJ, encoding an inverted box of genes for the λ phage repressor protein cI and the λ promoter pR (Figure 13).



Figure 13.Composition and Principle of the Red Light-Induced System

Experimental Approach

PCR technology was employed to generate fragments for the construction of plasmid pRED-GFP, with details presented in Figure 14a. 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 14c.​

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 14d. After cultivation, the correct plasmid was extracted and transformed into Escherichia coli BL21, as shown in Figure 14b. Through this entire procedure, we successfully obtained bacterial strains harboring the correct plasmid pRED.



Figure 14. 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

Characterization/Measurement

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 15). Only under red light induction did the strains exhibit significantly high fluorescence intensity, confirming the feasibility of our strain modifications.



Figure 15. Fluorescence intensity of the four strains under dark and red light conditions.