Part:BBa_K5526006

Plldr(New)-antiPD-L1

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 415
  Illegal AgeI site found at 754
  
• 1000
  COMPATIBLE WITH RFC[1000]

Composite Part: BBa_K5526006 (Plldr(new)-antiPD-L1)

Construction Design

In the plasmid Plldr(new)-antiPD-L1 (BBa_K5526006) we constructed, we combined Plldr-new (BBa_K5526001), antiPD-L1 (BBa_K5526008), and pUC57-mini (BBa_K3983004) to form Plldr(new)-antiPD-L1 (plactate2-antiPD-L1). Plldr-new is more accurate and will not be inhibited under low oxygen concentrations. AntiPD-L1 encodes an immunomodulatory protein that activates the immune system and targets tumor cells. pUC57-mini serves as the backbone of the plasmid. Plldr(new)-antiPD-L1 is activated in high lactic acid environments, typical of tumor areas, and produces an immunomodulatory drug that is more precise and effective under these conditions.

Figure 1. The plasmid map of Plldr(new)-antiPD-L1

Engineering Principle

In this project, we engineered the Plldr(new)-antiPD-L1 plasmid by combining Plldr-new, antiPD-L1, and pUC57-mini, resulting in an optimized plasmid for controlled drug production in high lactic acid environments such as tumor cells.

Experimental Approach

We applied PCR on the genes antiPD-L1 (372bp) and pUC57-plldr-new (3800bp) and confirmed the length of the PCR products through agarose gel electrophoresis, as shown in Figure 2. The results show that pUC57-plldr-new (plactate 2) has a length of 3800bp, and antiPD-L1 has a length of 372bp.

Figure 2. The identification of PCR production by agarose gel electrophoresis. Left: The graph shows that plactate 2 has a length of 3800 bp. Right: The graph shows that p2-antiPD-L1 has a length of 372bp.

We used homologous recombination to combine antiPD-L1 with the new Plldr promoter, forming Plldr(new)-antiPD-L1. After heat shock transformation of DH5α cells, bacterial colonies grew on the Amp+ medium, indicating successful plasmid uptake. Colony PCR was performed to confirm the presence of the correct plasmid, as seen in Figure 3. The sequencing results confirmed that the plasmid had the correct sequence and no mutations.

Figure 3. PCR identification of plactate2-antiPD-L1 plasmid. A: The length of p2-antiPD-L1 is 372bp. B: The flora growing in a petri dish. C: Sequencing results.

Characterization/Measurement

We used alkaline lysis to extract plasmids (plactate2-antiPD-L1) from bacterial cultures and performed PCR verification on EcN1917 transformants. The results, shown in Figure 4, confirmed that the plasmids were successfully transformed into EcN1917.

Figure 4. PCR identification of EcN1917 transformants. A: PCR production of the plactate2-Anti-PD-L1 transformants by agarose gel electrophoresis. B: Flora growing in a petri dish.

We tested protein expression under different OD values (0.3, 0.6, 0.8, 1.0) and lactic acid concentrations (0mM, 2mM, 5mM, 10mM). The protein concentration was highest when OD600 was 0.6 and lactic acid concentration was 5mM. We used SDS-PAGE to verify the proteins we obtained.

Figure 5. The effects of bacterial concentration and lactic acid concentration on the expression of plactate2-antiPD-L1.

Other Test

We expanded the culture and induced protein expression using 5 mM lactic acid in EcN1917. As shown in Figure 6, the Anti-PD-L1 protein was successfully expressed, with an expected size of about 24kDa. This confirmed that Anti-PD-L1 protein was expressed as intended. In the future, we plan to study the tumor inhibition effects of the constructed probiotics in a tumor environment.

Figure 6. Detection of Anti-PD-L1 protein expression by SDS-PAGE.

Summary

EcN drug molecular delivery carriers offer good compliance, long-lasting efficacy, and therapeutic precision, but several challenges remain before clinical application. Future animal experiments will verify the tumor inhibition effects of our engineered probiotics in vivo. More research is required to improve EcN strains for safer and more effective use in clinical treatments. Thus, much work remains to achieve precision tumor treatment using EcN drug delivery vectors.