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.

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.

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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.

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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.

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

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.

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.

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.

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.

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.

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.

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.

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

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

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.