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 [5]; 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.

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 characterization 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 Journal of Investigative Dermatology 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.

[9] iGEM 2018 Pasteur Paris. Available at: https://2018.igem.org/Team:Pasteur_Paris/Fighting

[10] Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021;596:583-9. https://doi.org/10.1038/s41586-021-03819-2.

[11] Varadi M, Anyango S, Deshpande M, Nair S, Natassia C, Yordanova G, et al. AlphaFold Protein Structure Database: Massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res 2022;50:D439-44. https://doi.org/10.1093/nar/gkab1061

[12] Cuong Vuong, Christiane Gerke, Greg A. Somerville, Elizabeth R. Fischer, Michael Otto, Quorum-Sensing Control of Biofilm Factors in Staphylococcus epidermidis, The Journal of Infectious Diseases, Volume 188, Issue 5, 1 September 2003, Pages 706-718, https://doi.org/10.1086/377239