Part:BBa_K5526002

Plldr-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 BsaI.rc site found at 335
  Illegal SapI.rc site found at 354
  

New Composite Part: BBa_K5526002 (Plldr_sfGFP)

Construction Design

In the plasmid Plldr_sfGFP (referred to as plactate1-sfGFP), 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 activated by a high lactic acid concentration, typical of tumor areas. sfGFP will be transcribed and form fluorescent protein. pUC57 serves as the skeleton of the plasmid. Additionally, Amp+ ensures that only EcN1917 with the correct plasmid will grow. Plactate1-sfGFP is a plasmid that can be activated and produce fluorescent proteins when exposed to high lactic acid concentrations.

Figure 1. The plasmid map of Plldr_sfGFP

Engineering Principle

We applied PCR on the genes sfGFP(750bp) and pUC57- Plldr (3150bp). Agarose gel electrophoresis was used to check the length of our PCR product to ensure success. The results showed that pUC57- Plldr (plactate1) had a length of 3150 bp, and sfGFP had a length of 750 bp (Figure 2).

Figure 2. The identification of PCR production by agarose gel electrophoresis. Left: pUC57-Plldr (3150 bp). Right: p1-sfGFP (750bp).

Experimental Approach

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 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 plactate1-sfGFP plasmid. A: p1-sfGFP (750bp). B: Bacterial colonies in petri dish. C: Gene sequencing results.

Characterization/Measurement

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

1. Fluorescence Microscope

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 4).

Figure 4. Microscopic images of bacteria under white light and fluorescence. sfGFP reaches the highest fluorescence intensity at 5mM lactic acid concentration.

2. Fluorescent Microplate Reader

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. Highest intensity is at 5mM 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.