In our contribution section, we decided to delve deeper into the investigation of the commonly used reporter gene, Red Fluorescent Protein (RFP). Red Fluorescent Proteins (RFPs) possess a strong absorption of light at the 600 nm wavelength, leading to a misrepresentation of cell optical density and consequently an underestimation of single-cell fluorescence. Thus, we selected BBa_J04450 from the iGEM part repository and used pSB1C3 as the vector to transform it into E. coli DH5α for expression, aiming to explore the influence of different light wavelengths on the deviation in measuring Red Fluorescent Protein (RFP) abundance in cells.

1. We activated BBa_J04450 obtained from the kit and transformed it into E. coli DH5α, followed by overnight incubation at 37℃. The subsequent day, we chose 4 colonies and cultured them in 5 mL LB medium for approximately 12 hours. This genetic segment was expressed within the plasmid pSB1C3; consequently, both our LB agar plates and culture media were supplemented with chloramphenicol (50 μg/mL).

2. Used XbaⅠ and SpeⅠ enzymes to construct the empty vector pSB1C3.

3. The pSB1C3 plasmid was transformed into E. coli DH5α, followed by overnight incubation at 37℃. The next day, four colonies were selected and cultured in 5 mL LB medium for approximately 12 hours.

4. We prepared six fresh 50 mL LB media. Subsequently, we introduced overnight cultures of E. coli carrying pSB1C3-J04450 and pSB1C3, respectively, into these media. The initial three media were inoculated with E. coli containing pSB1C3, while the latter were E. coli containing pSB1C3-J04450. These cultures were cultivated for sixteen hours.

5. After sampling, we promptly measured the data using an microplate reader (Tecan Spark) and conducted subsequent analysis.

Among commonly used fluorescent proteins, the excitation wavelength for mRFP is situated around 585 nm (Fig. 2). Consequently, it exhibits a prominent absorption peak at 600 nm (Fig. 2), a standard wavelength used for measuring bacterial culture optical density (OD). This phenomenon leads to a significant overestimation of data when quantifying the abundance of bacteria expressing red fluorescent protein.

Initially, we measured the fluorescence levels of E. coli. Notably, the E. coli carrying pSB1C3-J04450 exhibited apronounced shift towards red fluorescence in the later stages. In the early stages, E. coli carrying pSB1C3-J04450 displayed fluorescence levels similar to those without it. However, after 12 hours, a substantial increase was observed, significantly surpassing the fluorescence levels of the E. coli carrying pSB1C3 (Fig. 3). This indicates the density of our experimental strain, which exhibits a distinct disparity compared to E. coli carrying pSB1C3.

We measured the OD values of the bacteria, revealing distinct differences between E. coli strains carrying pSB1C3-J04450 and those carrying pSB1C3 at various wavelengths. Specifically, data obtained under 585 nm wavelength exhibited the most significant disparities between the two E. coli strains, while data acquired under 700 nm wavelength displayed less obvious distinctions (Fig. 4a-d). The increased disparity at 585 nm can be attributed to the absorption peak of mRFP occurring near this wavelength, thus making the data differences between the two strains more pronounced. Thus, the influence of RFP can be disregarded at the two longer wavelengths.

Furthermore, to gain further insight into the impact of RFP on E. coli OD values, we conducted deviation analyses relative to the baseline at 700 nm and performed linear function fitting for the data obtained at the other three wavelengths. It is evident that the data acquired at 660 nm exhibited minimal deviation, while the other two shorter wavelengths displayed a noticeable upward trend (Fig. 4e). At the 12-hour mark, the OD values obtained at 600 nm were nearly 15% higher than those unaffected by RFP. This illustrates the impact of mRFP expression on the measurement of bacterial density under wavelength at 600 nm. This serves as a valuable reference for future teams.

Hecht A, Endy D, Salit M, et al. When wavelengths collide: bias in cell abundance measurements due to expressed fluorescent proteins[J]. ACS synthetic biology, 2016, 5(9): 1024-1027.

Campbell R E, Tour O, Palmer A E, et al. A monomeric red fluorescent protein[J]. Proceedings of the National Academy of Sciences, 2002, 99(12): 7877-7882.