Part:BBa_K4345003

mPapaya

mPapaya is a constitutively fluorescent protein with an excitation wavelength of 530 nm and an emission wavelength of 541 nm. mPapaya is derived from Zoanthus sp. The excitation and emission spectra are presented below.

This picture was obtained from fpbase.org [1].

Exeter iGEM 2023

Usage and Biology

Whilst waiting for the parts designed for our project to be synthesised, we wanted to get into the lab and familiarise ourselves with the equipment and techniques. Our supervisor suggested we practised transforming, growing and expressing proteins in bacteria with fluorescent proteins. These are relatively non-toxic to bacteria and easy to visualise which allows for conformation of transformation and protein expression. During training on the imaging flow cytometer, we noticed some unusual results. These are presented below and are our bronze medal contribution to the iGEM registry.

Expression of sfGFP [2], mCherry [3] and mPapaya [4] was under control of the strong constitutive promoter BBa_J23100, combined with the strong RBS BBa_B0034 and terminated by the double terminator BBa_B0015. The gene was carried on a medium copy number plasmid with ampicillin resistance and transformed into E. coli DH5𝛼. 5 mL cultures were grown at 37 C, 200 rpm for 18 h. Cell morphology was investigated using an Image Stream Mark II (Amnis-Luminex Corp.) Imaging Flow Cytometer configured with Bright Field (white light), Side-Scatter (785 nm) and either GFP (excitation laser 488 nm, emission 533/55 nm), RFP (excitation laser 592 nm, emission 610/30 nm) or YFP (excitation laser 488 nm, emission 702/85 nm).

Imaging Flow Cytometry data was graphically analysed using specialised software [5]. The software can graphically represent the distribution of labelled cell populations, which can then be gated (selecting the region of cells to be analysed). Only the main population of cells were gated, with the outliers and speed beads used to calibrate the Flow Cytometer being disregarded.

Characterisation

Imaging Flow Cytometry collects two types of optical information: forward scatter (FSC) and side scatter (SSC) [5]. The main population of cells to be analysed was gated from a scatter plot of FSC against SSC (Figure 1). Figure 1: Scatter graphs of forward scatter against side scatter, with the gated regions selected. From left to right: mCherry, mPapaya, sfGFP.

Cell Length

The images in Figure 2 show that there is a distinct difference in cell length between cells expressing mPapaya in comparison to cells expressing mCherry and sfGFP. Cells expressing mCherry (Figure 2a) and sfGFP (Figure 2c) appear spherical and rod-shaped, which is a healthy shape for E. coli [6]; whereas cells expressing mPapaya1 appear much longer and 'noodly', (Figure 2b). The geometric mean of cell length for cells expressing mPapaya1 was larger (8.005 µm) than in cells expressing mCherry (2.544 µm) and cells expressing sfGFP (3.862 µm) (see Figure 3). Figure 2: Flow Cytometry images of the cells expressing fluorescent proteins, displaying how the cells expressing mPapaya have different morphology in comparison to the others. The first column is the brightfield, the second column is the forward scatter, and the third column is the side scatter. a) Cells expressing mCherry. b) Cells expressing mPapaya1. c) Cells expressing sfGFP.

Figure 3: From left to right: mCherry, mPapaya, sfGFP. Histograms showing the distribution of cell length. The data shows that cells expressing mPapaya1 are on average longer (geometric mean=8.005 µm) than the other cells( mCherry geometric mean=2.544 µm, sfGFP geometric mean=3.862 µm.

Fluorescence Intensity

Due to differences in quantum yield and extinction coefficient (i.e. brightness) between the three proteins it is not possible to compare expression levels of mPapaya, sfGFP and mCherry without calibration. Furthermore, the practical brightness of FPs is determined by several factors, such as folding, maturation efficiency, pKa, as well as the optical properties of the imaging equipment, therefore molecular brightness may not reflect the actual brightness of a protein during an experiment [7]. However, it is possible to compare cell variability within a population of cells. The flow cytometric histograms in Figure 4 show normalised frequency against fluorescence intensity. The coefficient of variation values (CV, %) are 84 for mPapaya,1 76 for mCherry, and 57 for sfGFP, demonstrating that cultures expressing mPapaya displays the widest range of fluorescent intensity in comparison to cultures expressing sfGFP and mCherry. Figure 4: From left to right: mCherry, mPapaya, sfGFP. Histograms showing the distribution of cell fluorescence intensity, showing that mPapaya displays the widest range of fluorescence intensity. However, practical brightness cannot be compared between the cells without calibration first.

Discussion

Cell morphology is often characteristic of a particular bacterial species. E. coli cells for example are often reported as being rod-shaped 2 µm long and 1 µm diameter [6]. However, bacterial cell morphology can change in response to the stage of life cycle and environmental factors [8]. This was seen during our experiments where E. coli cells expression mPapaya were observed to be longer than cells expressing either sfGFP or mCherry (see Figure 2). This increased cell length which could be representative of unhealthy cells, may be the reason why there is a wider variability in fluorescence of individual cells in populations expressing mPapaya. We unfortunately did not have time to investigate this further, but we hope that contributing our observations to the iGEM registry will inspire future teams' experiments.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 267
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 267
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 267
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 267
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 267
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] mPapaya. (2022). FPBase. Retrieved July 7, 2022, from https://www.fpbase.org/protein/mpapaya/

[2] Hoi, H., Howe, E. S., Ding, Y., Zhang, W., Baird, M. A., Sell, B. R., Allen, J. R., Davidson, M. W. & Campbell, R. E. 2013. An engineered monomeric Zoanthus sp. yellow fluorescent protein. Chem. Biol., 20, 1296-304.

[3] Pedelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. 2006. Engineering and characterisation of a superfolder green fluorescent protein. Nat. Biotechnol., 24, 79-88.

[4] Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E. & Tsien, R. Y. 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol., 22, 1567-72.

[5] Jahan-Tigh, R. R., Ryan C., Obermoser G., Schwarzengerger K., 2012 Flow Cytometry. J. Invest. Dermatol. 132, DOI: 10.1038/jid.2012.282

[6]Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol., 166, 557-80.

[7] Shaner, N.C., Steinbach, Tsien, 2005 A guide to choosing fluorescent proteins, Nat. Methods ,2, 905-909 DOI: 10.1038/nmeth819

[8] Power, A. L., Barber, D. G., Groenhof, S. R. M., Wagley, S., Liu, P., Parker, D. A. & Love, J. 2021. The Application of Imaging Flow Cytometry for Characterisation and Quantification of Bacterial Phenotypes. Front. Cell Infect. Microbiol., 11, 716592.