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

<!DOCTYPE html> Plldr_sfGFP (BBa_K5526002) Documentation

New Basic 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.

Plasmid map of Plldr_sfGFP — 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 used homologous recombination to combine sfGFP with the Plldr promoter, forming Plldr-sfGFP (plactate1-sfGFP). Heat shock conversion was performed to facilitate the uptake of plasmids by BL21(DE3) cells, which were then grown on an Amp+ medium. Bacterial colonies grew on the petri dishes, indicating successful plasmid uptake. Colony PCR was performed directly from the colonies to further confirm the presence of the desired plasmid (Figure 3). Sequencing confirmed the plasmids were correct, with 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

The fluorescence microscope was used to visually observe the lightness of sfGFP at different lactic acid concentrations. The results showed that the fluorescence intensity reached the highest at 5mM lactic acid concentration (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

A quantitative test using the fluorescent microplate reader provided precise numerical data on the fluorescence emitted by the cells. Data analysis confirmed that the fluorescence intensity of sfGFP was highest at a 5mM lactic acid 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.