Part:BBa_K5526003

Plldr(New)-sfGFP

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 400
  

Construction Design

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, a 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+ 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 to low oxygen concentration, like what the original one did instead.

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

Engineering Principle

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).

Experimental Approach

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 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 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 3 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 3. 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.

Characterization/Measurement

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 4).

Figure 4. 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

Using the fluorescent microplate reader, we then applied a quantitative test to the sfGFP. 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 5).

Figure 5. 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.