In the plasmid Plldr_sfGFP (referred to as plactate1-sfGFP) we constructed, we combined Plldr(BBa_K822000), sfGFP(BBa_K4716993), and pUC57-mini(BBa_K3983004) together to form Plldr-sfGFP(BBa_K822002).

Plldr is a lactic acid promoter that will be activated under a high lactic acid concentration, which is a trait of tumor areas. sfGFP will be transcribed and form fluorescence protein. pUC57 is the skeleton of the plasmid. We’d also add Amp+ into it to ensure only the EcN1917 with the correct plasmid will grow. plactate1-sfGFP is the plasmid that can be activated and produce fluorescent proteins by a high lactic acid concentration.

Figure 2. The plasmid map of Plldr_sfGFP

We applied PCR on the genes sfGFP(750bp) and pUC57- Plldr (3150bp); we used agarose gel electrophoresis to check the length of our PCR production to ensure we succeeded. The results showed that the pUC57- Plldr(plactate1) had a length of 3150 bp, and the sfGFP had a length of 750 bp (Figure 3).

Figure 3. The identification of PCR production by agarose gel electrophoresis
Left: The graph shows that plactate 1 has a length of 3150 bp.
Right: the graph shows p1-sfGFP has a length of 750bp.

We first used homologous recombination to combine sfGFP with the lldr promoter, forming the Plldr-sfGFP (plactate1-sfGFP) construct. We then performed a heat shock conversion to make BL21(DE3) cells sensitive to frequent changes in temperature, alternating between high and low temperatures to facilitate the uptake of plasmids of BL21(DE3). After heat shock, we injected the plasmids into BL21(DE3) cells and grew them on an Amp+ medium, ensuring that only bacteria containing the plasmids would survive. As expected, bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. To further confirm the presence of the desired plasmid, we performed a colony PCR directly from the colonies on the plate. This allowed us to amplify the specific region of the plasmid containing the Plldr-sfGFP(plactate1-sfGFP) construct. Figure 4 shows the PCR results were positive, indicating that the colonies contained the correct plasmid. Finally, we recycled the plasmids and sent them for sequencing at a bio company to ensure the correct sequence. The sequencing results confirmed that the plasmids were indeed the ones we wanted, with the correct sequence and no mutations.

Figure 4. PCR identification of plactate1-sfGFP plasmid.
A. The graph shows the length of p1-sfGFP is 750bp.
B. The graph shows the flora growing in a petri dish.
C. The graph shows the sequencing measured by the bio company.

We analyzed the fluorescence intensity of sfGFP produced using two different approaches:

Fluorescence microscope: We first used the fluorescence microscope to test the lightness of the sfGFP. This is a qualitative test to visually observe under what concentration of lactic acid the lightness of sfGFP will reach the highest. The microscope provided qualitative data, showing that the fluorescence intensity reaches the highest when the lactic acid concentration is 5mM, indicating that the Plldr-sfGFP (plactate1-sfGFP) construct was functioning as intended (Figure 5).

Figure 5. The microscopic images of bacteria under white light and fluorescence.
The graph shows that sfGFP reaches the highest fluorescence intensity at 5mM of lactic acid concentration.

Fluorescent Microplate Reader: We then applied a quantitative test to the sfGFP using the fluorescent microplate reader. The microplate reader provided precise numerical data on the fluorescence emitted by the cells; we analyzed the data and drew a graph based on it. From this analysis, we concluded that the fluorescence intensity of sfGFP was highest at a lactic acid concentration of 5mM, confirming the optimal response of the construct to this concentration (Figure 6).

Figure 6. Bacterial fluorescence intensity at different lactate concentrations. The graph we drew after analyzing the data from the fluorescent microplate reader also shows the highest light intensity at 5mM of lactic acid concentration.

This plasmid was constructed from a comparison with Plldr(new)-sfGFP to show whether the improvement on the new Plldr is functional. The experiment would be successful if the Plldr(new)-sfGFP got a higher light intensity than Plldr-sfGFP.

In the plasmid Plldr(new)-sfGFP (BBa_K5526003) we constructed, we combined Plldr-new (BBa_K5526001), sfGFP (BBa_K4716993), and pUC57-mini (BBa_K3983004) together to form Plldr(new)-sfGFP (plactate2-sfGFP).

Plldr(new) is a lactic acid promoter that will be activated under a high lactic acid concentration, which is the trait of tumor areas. It gets several improvements to the original promoter. It is more accurate and will not get inhibited within low oxygen concentrations. sfGFP will be transcribed and form fluorescence protein. pUC57 is the skeleton of the plasmid. We’d also add Amp+ into it to ensure only the EcN1917 with the correct plasmid will grow. Plldr(new)-sfGFP is the plasmid that can be activated and produce fluorescent proteins by a high lactic acid concentration. Plldr(new)-sfGFP is more sensitive and precise when increasing lactic acid concentration. In addition, it will not be limited within low oxygen concentration, like what the original one did instead.

Figure 7. The plasmid map of Plldr(new)-sfGFP

We applied PCR on the genes sfGFP(750bp) and pUC57- Plldr (new)(3800bp); we used agarose gel electrophoresis to check the length of our PCR production to ensure we succeeded. The result is that the plactate 2 got a length of 3800bp, and the sfGFP got a length of 750bp.

Figure 8. 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 p2-sfGFP has a length of 750bp.

We first used homologous recombination to combine sfGFP with the new lldr promoter, forming the Plldr(new)-sfGFP (plactate 2-sfGFP) construct. We then performed a heat shock conversion to make BL21(DE3) cells sensitive to frequent changes in temperature, alternating between high and low temperatures to facilitate the uptake of plasmids of BL21(DE3). After heat shock, we injected the plasmids into BL21(DE3) cells and grew them on an Amp+ medium, ensuring that only bacteria containing the plasmids would survive. As expected, bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. To further confirm the presence of the desired plasmid, we performed a colony PCR directly from the colonies on the plate. This allowed us to amplify the specific region of the plasmid containing the Plldr(new)-sfGFP (plactate 2-sfGFP) construct. Figure 9 shows the PCR results were positive, indicating that the colonies contained the correct plasmid. Finally, we recycled the plasmids and sent them for sequencing at a bio company to ensure the correct sequence. The sequencing results confirmed that the plasmids were indeed the ones we wanted, with the correct sequence and no mutations.

Figure 9. PCR identification of plactate2-sfGFP plasmid.
A. The graph shows the length of p2-sfGFP is 750bp.
B. The graph shows the flora growing in a petri dish.
C. The graph shows the sequencing measured by the bio company.

We analyzed the fluorescence intensity of sfGFP produced using two different approaches:

Fluorescence microscope: We first used the fluorescence microscope to test the lightness of the sfGFP. This is a qualitative test to visually observe under what concentration of lactic acid the lightness of sfGFP will reach the highest. The microscope provided qualitative data, showing that the fluorescence intensity reaches the highest when the lactic acid concentration is 5mM, indicating that the Plldr(new)-sfGFP (plactate 2-sfGFP) construct was functioning as intended (Figure 10).

Figure 10. The microscopic images of bacteria under white light and fluorescence.
The graph shows that the sfGFP reaches the highest fluorescence intensity at 5 mM for lactic acid concentration.

Fluorescent Microplate Reader: We then applied a quantitative test to the sfGFP using the fluorescent microplate reader. The microplate reader provided precise numerical data on the fluorescence emitted by the cells; we analyzed the data and drew a graph based on it. From this analysis, we concluded that the fluorescence intensity of sfGFP was highest at a lactic acid concentration of 5mM, confirming the optimal response of the construct to this concentration (Figure 11).

Figure 11. Bacterial fluorescence intensity at different lactate concentrations. The graph we drew after analyzing the data from the fluorescent microplate reader also shows the highest light intensity at 5mM of lactic acid concentration.

This plasmid has been constructed from a comparison with Plldr-sfGFP to show whether the improvement on the new Plldr is functional. The experiment would be successful if the Plldr(new)-sfGFP got a higher light intensity than Plldr-sfGFP.

In the plasmid plactate1-antiPD-L1(BBa_K5526004) we constructed, we combined Plldr(BBa_K822000), antiPD-L1(BBa_K5526008), and pUC57-mini(BBa_K3983004) together to form Plldr-antiPD-L1(plactate 1- antiPD-L1).

Plldr is a lactic acid promoter that will be activated under a high lactic acid concentration. antiPD-L1 is a set gene that encodes an immunomodulatory protein, which can activate the human body’s immune system and kill the tumor cells. pUC57 is the skeleton of the plasmid. Plldr-antiPD-L1 is the plasmid that will be activated and produce an immunomodulatory drug by high lactic acid concentration.

Figure 12. The plasmid map of Plldr- anti PD-L1

We applied PCR on the genes antiPD-L1(372bp) and pUC57-plldr(3150bp); we used agarose gel electrophoresis to check the length of our PCR production to ensure we succeeded. The result is that the pUC57-plldr (plactate 1) got a length of 3150bp, and the antiPD-L1 got a length of 372bp.

Figure 13. The identification of PCR production by agarose gel electrophoresis
Left: The graph shows that plactate 1 has a length of 3150 bp.
Right: The graph shows that antiPD-L1 has a length of 372bp.

We first used homologous recombination to combine anti PD-L1 with the lldr promoter, forming the Plldr-anti PD-L1. We then performed a heat shock conversion to make DH5α cells sensitive to frequent changes in temperature, alternating between high and low temperatures to facilitate the uptake of plasmids of DH5α. After heat shock, we injected the plasmids into DH5α cells and grew them on an Amp+ medium, ensuring that only bacteria containing the plasmids would survive. As expected, bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. To further confirm the presence of the desired plasmid, we performed a colony PCR directly from the colonies on the plate. This allowed us to amplify the specific region of the plasmid containing the Plldr-antiPD-L1(plactate 1- antiPD-L1) construct. Figure 14 shows the PCR results were positive, indicating that the colonies contained the correct plasmid. Finally, we recycled the plasmids and sent them for sequencing at a bio company to ensure the correct sequence. The sequencing results confirmed that the plasmids were indeed the ones we wanted, with the correct sequence and no mutations.

Figure 14. PCR identification of plactate1-antiPD-L1 plasmid.
A. The graph shows the length of p1-antiPD-L1 is 372bp.
The graph shows the flora growing in a petri dish.
C. The graph shows the sequencing measured by the bio company.

We used alkaline lysis to extract plasmids (plactate1-Anti-PD-L1) from bacterial cultures. Next, we converted the recombinant plasmid to EcN1917 competent by heat shock transformation. PCR was used to verify whether the plasmid was transformed into EcN1917. The results are shown in Figure 15-A. The colony PCR results were detected by agarose gel electrophoresis, and the amplified band was 550bp, which was consistent with the target band, indicating that the four plasmids were successfully transformed into EcN1917.

Figure 15. PCR identification of EcN1917 transformants.
A. The PCR production of the plactate1-Anti-PD-L1 transformants by agarose gel electrophoresis.
B. The graph shows the flora growing in a petri dish.

We grow the bacteria holding the plasmids under different OD values (0.3, 0.6, 0.8,1.0) and lactic acid concentrations (0mM, 2mM, 5mM, 10mM). We used the nanodrop to detect protein concentration and created a graph of four different bacteria’s protein expression. We conclude that the protein concentration is highest when OD600 equals 0.6, and the lactic acid concentration is 5mM. We used SDS-PAGE to ensure we got the proteins we wanted.

Figure 16. The effects of bacterial concentration and lactic acid concentration on the expression of plactate1-anti PD-L1.

This plasmid Plldr-antiPD-L1(plactate1-antiPD-L1) was constructed to form a comparison with Plldr(new)-antiPD-L1. If the concentration of proteins produced by the Plldr(new)-antiPD-L1 is higher than Plldr-antiPD-L1(plactate1-antiPD-L1), this can show that improving the new Plldr is useful.

In the plasmid Plldr(new)-antiPD-L1 (BBa_K5526006) we construct, we combined Plldr-new (BBa_K5526001), antiPD-L1 (BBa_K5526008), and pUC57-mini (BBa_K3983004) together to form Plldr(new)-antiPD-L1 (plactate2-antiPD-L1).

Plldr-new gets several improvements to the original promoter. It is more accurate and will not get inhibited within low oxygen concentrations. antiPD-L1 is a set gene that encodes an immunomodulatory protein, which can activate the human body’s immune system and kill the tumor cells. pUC57-mini is the skeleton of the plasmid. Plldr(new)-antiPD-L1 (plactate2-antiPD-L1) is the plasmid that will be activated and produce an immunomodulatory drug by high lactic acid concentration. It is more sensitive and precise when increasing lactic acid concentration. In addition, it will not be limited within low oxygen concentration, like what the original one did instead.

Figure 17. The plasmid map of Plldr(new)-anti PD-L1

We applied PCR on the genes antiPD-L1(372bp) and pUC57-plldr-new(3800bp). We used agarose gel electrophoresis to check the length of our PCR production to ensure we succeeded. The result is that the pUC57-plldr-new (plactate 2) got a length of 3800bp, and the antiPD-L1 got a length of 372bp.

Figure 18. 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 first used homologous recombination to combine antiPD-L1 with the new lldr promoter, forming the Plldr(new)-antiPD-L1 (plactate2-antiPD-L1). We then performed a heat shock conversion to make DH5α cells sensitive to frequent changes in temperature, alternating between high and low temperatures to facilitate the uptake of plasmids of DH5α. After heat shock, we injected the plasmids into DH5α cells and grew them on an Amp+ medium, ensuring that only bacteria containing the plasmids would survive. As expected, bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. To further confirm the presence of the desired plasmid, we performed a colony PCR directly from the colonies on the plate. This allowed us to amplify the specific region of the plasmid containing the plactate2-antiPD-L1 construct. Figure 18 shows the PCR results were positive, indicating that the colonies contained the correct plasmid. Finally, we recycled the plasmids and sent them for sequencing at a bio company to ensure the correct sequence. The sequencing results confirmed that the plasmids were indeed the ones we wanted, with the correct sequence and no mutations.

Figure 19. PCR identification of plactate2-antiPD-L1 plasmid.
A. The graph shows the length of p2-antiPD-L1 is 372bp.
B. The graph shows the flora growing in a petri dish.
C. The graph shows the sequencing measured by the bio company.

We used alkaline lysis to extract plasmids (plactate2-Anti-PD-L1) from bacterial cultures. Next, we converted the recombinant plasmid to EcN1917 competent by heat shock transformation. PCR was used to verify whether the plasmid was transformed into EcN1917. The results are shown in Figure 20-A. The colony PCR results were detected by agarose gel electrophoresis, and the amplified band was 550bp, which was consistent with the target band, indicating that the four plasmids were successfully transformed into EcN1917.

Figure 20. PCR identification of EcN1917 transformants.
A. The PCR production of the plactate2-Anti-PD-L1 transformants by agarose gel electrophoresis.
B. The graph shows the flora growing in a petri dish.

We grow the bacteria holding the plasmids under different OD values (0.3, 0.6, 0.8,1.0) and lactic acid concentrations (0mM, 2mM, 5mM, 10mM). We used the nanodrop to detect protein concentration and created a graph of four different bacteria’s protein expression. We conclude that the protein concentration is highest when OD600 equals 0.6, and the lactic acid concentration is 5mM. We used SDS-PAGE to ensure we got the proteins we wanted.

Figure 21. The effects of bacterial concentration and lactic acid concentration on the expression of plactate2-antiPD-L1.

This plasmid was constructed from a comparison of Plldr-antiPD-L1. If the concentration of proteins produced by the Plldr(new)-antiPD-L1(plactate2-antiPD-L1) is higher than Plldr-antiPD-L1, this can show that improving the new Plldr is useful.

In the plasmid Plldr-Azurin (BBa_K5526005) we constructed, we combined Plldr (BBa_K822000), Azurin (BBa_K5526000), and pUC57-mini (BBa_K3983004) to form Plldr-Azurin (plactate 1-Azurin).

Plldr is a lactic acid promoter that will be activated under a high lactic acid concentration. Azurin is a gene that encodes a chemical drug, which can release some medicines and substances to kill the tumor cells. pUC57-mini is the skeleton of the plasmid. Plldr-Azurin is the plasmid that will be activated and produce a toxic protein with a high lactic acid concentration.

Figure 22. The plasmid map of Plldr- Azurin.

We applied PCR on the genes Azurin(444bp) and pUC57-plldr (3150bp); we used agarose gel electrophoresis to check the length of our PCR production to ensure we succeeded. The result shows that the pUC57-plldr (plactate 1) has a length of 3150bp, and the Azurin has a length of 444bp.

Figure 23. The identification of PCR production by agarose gel electrophoresis
Left: The graph shows that plactate 1 has a length of 3150 bp.
Right: The graph shows p1-Azurin has a length of 444bp.

We first used homologous recombination to combine Azurin with the lldr promoter, forming the Plldr-Azurin (plactate1-Azurin). We then performed a heat shock conversion to make DH5α cells sensitive to frequent changes in temperature, alternating between high and low temperatures to facilitate the uptake of plasmids of DH5α. After heat shock, we injected the plasmids into DH5α cells and grew them on an Amp+ medium, ensuring that only bacteria containing the plasmids would survive. As expected, bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. To further confirm the presence of the desired plasmid, we performed a colony PCR directly from the colonies on the plate. This allowed us to amplify the specific region of the plasmid containing the Plldr-Azurin (plactate1-Azurin) construct. Figure 22 shows the PCR results were positive, indicating that the colonies contained the correct plasmid. Finally, we recycled the plasmids and sent them for sequencing at a bio company to ensure the correct sequence. The sequencing results confirmed that the plasmids were indeed the ones we wanted, with the correct sequence and no mutations.

Figure 24. PCR identification of plactate1-Azurin plasmid.
A. The graph shows the length of p1-Azurin is 444bp.
The graph shows the flora growing in a petri dish.
C. The graph shows the sequencing measured by the bio company.

We used alkaline lysis to extract plasmids (plactate1-Azurin) from bacterial cultures. Next, we converted the recombinant plasmid to EcN1917 competent by heat shock transformation. PCR was used to verify whether the plasmid was transformed into EcN1917. The results are shown in Figure 25-A. The colony PCR results were detected by agarose gel electrophoresis, and the amplified band was 550bp, which was consistent with the target band, indicating that the four plasmids were successfully transformed into EcN1917.

Figure 25. PCR identification of EcN1917 transformants.
A. The PCR production of the plactate1-Azurin transformants by agarose gel electrophoresis.
B. The graph shows the flora growing in a petri dish.

We grow the bacteria holding the plasmids under different OD values (0.3, 0.6, 0.8,1.0) and lactic acid concentrations (0mM, 2mM, 5mM, 10mM). We used the nanodrop to detect protein concentration and created a graph of four different bacteria’s protein expression. We conclude that the protein concentration is highest when OD600 equals 0.6, and the lactic acid concentration is 5mM. We used SDS-PAGE to ensure we got the proteins we wanted.

Figure 26. The effects of bacterial concentration and lactic acid concentration on the expression of plactate1-Azurin.

This plasmid was constructed to compare Plldr(new)-Azurin with Plldr-Azurin(plactate1-Azurin). If the concentration of proteins produced by the Plldr(new)-Azurin is higher than that produced by Plldr-Azurin(plactate1-Azurin), it can show that improving the new Plldr is useful.

In the plasmid Plldr(new)-Azurin (BBa_K5526007) we construct, we combined Plldr-new (BBa_K5526001), Azurin (BBa_K5526000), and pUC57-mini (BBa_K3983004) together to form Plldr(new)-Azurin (plactate2- Azurin).

Plldr(new) gets several improvements to the original promoter. It is more accurate and will not get inhibited within low oxygen concentrations. Azurin is a gene that encodes a chemical drug, which can release some medicines and substances to kill the tumor cells. pUC57-mini is the skeleton of the plasmid. Plldr(new)-Azurin (plactate2- Azurin) is the plasmid that will be activated and produce a toxic protein by high lactic acid concentration. In addition, it will not be limited within low oxygen concentration, like what the original one did instead.

Figure 27. The plasmid map Plldr(new)-Azurin

We applied PCR on the genes Azurin(444bp) and the new pUC57-plldr (3800bp). We used agarose gel electrophoresis to check the length of our DNA pieces to ensure we succeeded. The result shows that the new pUC57-plldr has a length of 3800bp, and the Azurin has a length of 444bp.

Figure 28. 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-Azurin has a length of 444bp.

We first used homologous recombination to combine Azurin with the new pUC57-plldr promoter, forming the Plldr(new)-Azurin (plactate 2-Azurin). We then performed a heat shock conversion to make DH5α cells sensitive to frequent changes in temperature, alternating between high and low temperatures to facilitate the uptake of plasmids of DH5α. After heat shock, we injected the plasmids into DH5α cells and grew them on an Amp+ medium, ensuring that only bacteria containing the plasmids would survive. As expected, bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. To further confirm the presence of the desired plasmid, we performed a colony PCR directly from the colonies on the plate. This allowed us to amplify the specific region of the plasmid containing the Plldr(new)-Azurin (plactate 2-Azurin) construct. Figure 26 shows the PCR results were positive, indicating that the colonies contained the correct plasmid. Finally, we recycled the plasmids and sent them for sequencing at a bio company to ensure the correct sequence. The sequencing results confirmed that the plasmids were indeed the ones we wanted, with the correct sequence and no mutations.

Figure 29. PCR identification of plactate2-Azurin plasmid.
A. The graph shows the length of p2-Azurin is 444bp.
B. The graph shows the flora growing in a petri dish.
C. The graph shows the sequencing measured by the bio company.

We used alkaline lysis to extract plasmids (plactate2-Azurin) from bacterial cultures. Next, we converted the recombinant plasmid to EcN1917 competent by heat shock transformation. PCR was used to verify whether the plasmid was transformed into EcN1917. The results are shown in Figure 30-A. The colony PCR results were detected by agarose gel electrophoresis, and the amplified band was 550bp, which was consistent with the target band, indicating that the four plasmids were successfully transformed into EcN1917.

Figure 30. PCR identification of EcN1917 transformants.
A. The PCR production of the plactate2-Azurin transformants by agarose gel electrophoresis.
B. The graph shows the flora growing in a petri dish.

We grow the bacteria holding the plasmids under different OD values (0.3, 0.6, 0.8,1.0) and lactic acid concentrations (0mM, 2mM, 5mM, 10mM). We used the nanodrop to detect protein concentration and created a graph of four different bacteria’s protein expression. We conclude that the protein concentration is highest when OD600 equals 0.6, and the lactic acid concentration is 5mM. We used SDS-PAGE to ensure we got the proteins we wanted.

Figure 31. The effects of bacterial concentration and lactic acid concentration on the expression of plactate 2-Azurin.

This plasmid was constructed to compare Plldr(new)-Azurin (plactate 2-Azurin) with Plldr-Azurin. If the concentration of proteins produced by the Plldr(new)-Azurin (plactate 2-Azurin) is higher than that produced by Plldr-Azurin, it can show that improving the new Plldr is useful.