Part:BBa_K5422011:Experience

Objective

The goal of this experiment was to observe the functionality of the part construct by examining the co-expression of GFP and mCherry proteins in E. coli cells. We aimed to observe whether the construct could successfully drive the simultaneous expression of these two fluorescent proteins, providing insight into the stability and effectiveness of the genetic system in a bacterial environment.

Method

Cultures of E. coli cells containing the part construct were grown for 5 hours following the dilution of a starter culture at 1:100. The cells were observed under a fluorescence microscope. To ensure accurate and comprehensive results, six different fields of view (F.O.V.) were captured from the samples, reducing the risk of bias from focusing on a potentially atypical or anomalous region. For clarity and conciseness, two representative F.O.V.s were selected to present the results. These fields (F.O.V 1 and F.O.V 2) were chosen based on their clear expression of both GFP and mCherry.

During image analysis, the brightness and contrast of GFP fluorescence were adjusted using ImageJ software (version 1.54g, Java 1.8.0_345, 64-bit) to avoid over-saturation, ensuring that details in the merged images could be accurately interpreted. This adjustment was necessary to balance the strong GFP signal and prevent interference in the detection of mCherry.

Results

The microscopy results revealed the simultaneous expression of GFP and mCherry in E. coli cells. In both F.O.V 1 and F.O.V 2, GFP expression was clearly visible as green fluorescence (an and d), while mCherry expression appeared as red fluorescence (b and e). In the merged images (c and f), the overlap of the GFP and mCherry signals resulted in yellow fluorescence, indicating the co-localization and co-expression of both fluorescent proteins within the cells. This co-expression confirmed that the PcGMc construct successfully directed the simultaneous production of GFP and mCherry, with no observable variation between different fields of view.

Discussion

These results demonstrate that the part construct is functioning as intended, successfully driving the co-expression of GFP and mCherry in E. coli. The consistency of the fluorescence signals across multiple fields of view suggests that the construct is stable and effectively expressed throughout the bacterial population. Adjusting the brightness and contrast of the GFP signal using ImageJ allowed us to standardize the results and avoid over-saturation, ensuring that the fluorescence signals from both proteins could be clearly distinguished.