In our project, we have added some new parts to iGEM (Table 1). This includes a newly synthesized gene Azurin (BBa_K5526000), a Plldr (new) promoter (BBa_K5526001), and six expression vectors formed by two promoters and three genes: Plldr sfGFP (BBa_K5526002), Plldr(New)-sfGFP (BBa_K5526003), Plldr-antiPD-L1 (BBa_K5526004), Plldr-Azurin (BBa_K5526005), Plldr(New)-antiPD-L1 (BBa_K5526006), and Plldr(New)-Azurin (BBa_K5526007). This project designed a lactic acid induced expression system for engineered probiotics. In general, drug proteins are not expressed. Still, when the engineered bacteria enter the high lactic acid environment inside the tumor, the promoter is activated, then expressed and releases anti-tumor drug proteins.
| Parts Code | Parts Name | Type I | Type II |
|---|---|---|---|
| BBa_K5526000 | Azurin | basic part | Coding |
| BBa_K5526001 | Plldr(New) promoter | basic part | Regulatory |
| BBa_K5526002 | Plldr-sfGFP | composite part | Plasmid |
| BBa_K5526003 | Plldr(New)-sfGFP | composite part | Plasmid |
| BBa_K5526004 | Plldr-anti-PD-L1 | composite part | Plasmid |
| BBa_K5526005 | Plldr-Azurin | composite part | Plasmid |
| BBa_K5526006 | Plldr(New)-anti-PD-L1 | composite part | Plasmid |
| BBa_K5526007 | Plldr(New)-Azurin | composite part | Plasmid |
| BBa_K5526008 | Anti-PD-L1 | basic part | Coding |
1.1 Add a Basic Part, BBa_K5526000 (Azurin)
Length: 444bp
Source: Pseudomonas aeruginosa, synthesized.
Properties: Azurin is a cupredoxin protein isolated from Pseudomonas aeruginosa that can enter cancer cells and induce apoptosis. It interacts with specific molecules on the surface of cancer cells, disrupting intracellular signaling pathways. Azurin is primarily in the stages of basic research and preclinical trials and has not yet become a standard clinical treatment.
Figure 1 Gene map of Azurin
Length: 390bp
Source: Bacteria, synthesized
Properties: The Plldr promoter, derived from *Lactobacillus plantarum*, is a lactate-inducible promoter known for its ability to respond to the presence of lactic acid. It is a moderately strong promoter, offering controlled expression levels of target genes. Plldr's activity is influenced by lactate concentration, making it useful for applications where gene expression needs to be tightly regulated in response to metabolic changes.
Figure 2 Gene map of Plldr new promoter
Length: 372bp
Source: cell, synthesized
Properties: PD-L1 is a protein expressed on the surface of many tumor cells that allows tumor cells to evade the immune system by binding to the PD-1 receptor on T cells in the immune system. By blocking the interaction between PD-L1 and PD-1, the T cell-mediated immune response is reactivated, thus helping the immune system to recognize and destroy the tumor cells. Anti-PD-L1, an immune checkpoint inhibitor that blocks the recognition of PD-L1 indicated by tumor cells with T cells and enhances the immune effect on tumors, has been widely used in the clinical treatment of many types of cancer. Especially in the field of immunotherapy, it has shown remarkable therapeutic effects.
Figure 3 Gene map of Anti-PD-L1
2.1 Add New Composite Part, BBa_K5526002(Plldr-sfGFP) and BBa_K5526003 (new Plldr-sfFGP)
A. Plasmid construction
We use Plldr promoter (BBa_K822000), sfGFP(BBa_K4716993), and pUC57-mini(BBa_K3983004) to compose the Plldr-sfGFP. We use a new Plldr promoter (BBa_K5526001), sfGFP(BBa_K4716993), and pUC57-mini(BBa_K3983004) to compose the new Plldr-sfGFP.
We constructed Plldr-sfGFP (referred to as plactate1-sfGFP) and new Plldr-sfGFP (referred to as plactate2-sfGFP) by using homologous recombination. The sfGFP sequence was amplified by PCR, with a length of 750 bp. Figure 4 indicates that the band is consistent with the results. The plactate 1 and plactate 2 plasmids were used as a template for PCR amplification, resulting in a fragment of 3150bp and 3800bp. Figure 4 shows a band consistent with the target size, indicating successful amplification of sfGFP and linearization plasmid. After gel recovery, the Plldr fragment was obtained. At the same time, we use the same method to get the new Plldr fragment.
Figure 4 PCR amplification (left: plasmid with promoter. right: target gene)
The plasmids were transformed into E. coli Bl21. Figure 5 shows the presence of single colonies on the plate. We selected 4 clones for colony PCR. We selected 10 clones and sent them directly for sequencing. According to the results shown in Figure 5, the plactate1-sfGFP and plactate2-sfGFP were successfully ligated to the pUC57-mini vector without any apparent mutations, confirming the successful construction of the plactate1-sfGFP and plactate2-sfGFP plasmid.
Figure 5 A: colony PCR verification. B: flat colonies. C: sequence
B. Fluorescence detection
This study induced sfGFP gene expression using different concentrations of lactic acid and monitored the expression of green fluorescent protein (GFP) using a fluorescence microscope. The results indicated that at a concentration of 5 mM, the fluorescence intensity of GFP was the highest, establishing 5 mM as the optimal lactic acid concentration.
Figure 6 Expression of sfGFP fluorescent protein induced by different lactic acids
After determining the optimal lactic acid concentration, the fluorescence of sfGFP induced by different lactic acid concentrations was measured using Fluorescent Enzyme Labeler, with excitation at 470 nm and emission at 509 nm. The results showed that at a lactic acid concentration of 5 mM, the relative fluorescence intensity of new Plldr-sfGFP was higher than that of Plldr-sfGFP, indicating that the new Plldr promoter has more vigorous activity. Therefore, this study will incorporate the new Plldr promoter into subsequent research on tumor drugs.
Figure 7 The expression of sfGFP induced by different lactic acids
A. Plasmid construction
We use the Plldr promoter (BBa_K822000), Anti-PD-L1(BBa_K5526008), and pUC57-mini (BBa_K3983004) to compose the Plldr-antiPD-L1(BBa_K5526004). We use the new Plldr promoter (BBa_K5526001), Anti-PD-L1 (BBa_K5526008), and pUC57-mini (BBa_K3983004) to compose the new Plldr-antiPD-L1(BBa_K5526006).
Azurin is a cupredoxin protein isolated from Pseudomonas aeruginosa that can enter cancer cells and induce apoptosis. It interacts with specific molecules on the surface of cancer cells, disrupting intracellular signaling pathways. Azurin is primarily in the stages of basic research and preclinical trials and has not yet become a standard clinical treatment.
Anti-PD-L1 is a single-chain antibody targeting programmed death-ligand 1 (PD-L1). PD-L1 is a protein commonly expressed on the surface of many tumor cells, which inhibits T cell activity by binding to the PD-1 receptor on T cells in the immune system. This allows tumor cells to evade immune system attacks, thereby surviving and spreading. Anti-PD-L1 drugs bind to PD-L1, blocking its interaction with the PD-1 receptor, thereby reactivating the immune response mediated by T cells. This enables the immune system to recognize and attack tumor cells. Anti-PD-L1 drugs have been widely used as immune checkpoint inhibitors to immunize various cancers. They have shown significant therapeutic effects, particularly in some cancers such as melanoma and non-small cell lung cancer. The development and application of these drugs represent a significant advancement in cancer therapy, altering the treatment landscape for many cancers.
We constructed Plldr-antiPD-L1, new Plldr-antiPD-L1, Plldr-Azurin, and new Plldr-Azurin using homologous recombination. The Anti-PD-L1 sequence was amplified by PCR, with a length of 372 bp. We also amplified Azurin by PCR, with a length of 444 bp. Figure 8 indicates that the band is consistent with the results. The pUC57-mini plasmid was used as a template for PCR amplification, resulting in a fragment of 2665 bp for pUC57-mini. Figure 8 shows a band consistent with the target size, indicating successful amplification of target genes and linearizing the plasmid. After gel recovery, the Plldr-antiPD-L1, new Plldr-antiPD-L1, Plldr-Azurin, and new Plldr-Azurin fragments were obtained.
Figure 8 PCR amplification of plasmids and target genes
The plasmids were transformed into E. coli DH5α. Figure 9 shows the presence of single colonies on the plate. We selected colonies 1-10 and sent them directly for sequencing. According to the results shown in Figure 9, all the plasmids were successfully constructed without any apparent mutations, confirming the successful construction of the Plldr-antiPD-L1, new Plldr-antiPD-L1, Plldr-Azurin, and new Plldr-Azurin plasmids.
Figure 9 A. colony PCR verification; B. flat colonies; C. sequence
The successfully sequenced plasmids were transformed into Escherichia coli (EcN) Nissle 1917 using the heat shock. After overnight incubation, single colonies were picked and verified by colony PCR using specific primers, as shown in Figures 10 A and B. The results indicate that we successfully transformed the four plasmids, Plldr-antiPD-L1, new Plldr-antiPD-L1, Plldr-Azurin, and new Plldr-Azurin plasmids, into ECN. Subsequent experiments will involve testing their expression levels using different lactic acid concentrations.
Figure 10 A. colony PCR verification; B. flat colonies
This study used different lactic acid concentrations to induce the protein expression of anti-PD-L1 and Azurin. A protein concentration detector was used to detect the protein concentrations of anti-PD-L1 and Azurin under different lactic acid concentrations. The results showed that the protein concentrations of the Plldr promoter and the new Plldr promoter were the highest when the lactate concentration was 5 mM, just like in Figure 11. Therefore, it is best to determine the lactic acid concentration of 5 mM.
In addition, according to the results shown in Figure 11, we further compared the activity of two lactic acid promoters, Plldr and new Plldr. At the optimal lactic acid concentration of 5mM, the protein expression controlled by the new Plldr promoter achieved higher concentrations. Finally, we concluded that the new Plldr promoter has a more vital ability to initiate expression.
Figure 11 The effects of lactic acid concentration on the expression of recombinant proteins
SDS-PAGE was used to detect protein expression at a lactic acid induced concentration of 5mM. The experimental results are shown in Figure 12; the size of Azurin was 19kDa, and that of antiPD-L1 was 24kDa, consistent with the actual size, indicating that the protein was successfully expressed.
Figure 12 Detection of Azurin and Anti-PDL1 protein expression by SDS-PAGE
Several inducible engineered bacteria have been developed for tumor drug delivery. For example, the expression of cytolysin A (ClyA) and the immunomodulator flagellin subunit (FlaB) is induced by ara [1-3], and the promotion of tumor immunity by light- or ultrasound-induced TNF-α and IFN-γ, for example [4-5]. Externally induced gene loops suffer from non-specific induction (small molecules) or the inability to treat deep or located tumor tissue (light and ultrasound). In contrast, promoters that respond directly to the tumor microenvironment help to build a more brilliant gene loop for tumor killing. There are various types of inducibly expressed promoters in bacteria that can respond to the tumor microenvironment (lactic acid, PH, hypoxia) [6-7], and our use of tumor-responsive promoters for intelligent drug delivery will provide a novel strategy for tumor-targeted therapy.
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[6]Chien, T., Harimoto, T., Kepecs, B. et al. Enhancing the tropism of bacteria via genetically programmed biosensors. Nat Biomed Eng 6, 94–104 (2022).
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[7]Raman, V., Van Dessel, N., Hall, C.L. et al. Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases. Nat Commun 12, 6116 (2021).