Data from the paper "Self-cleaving peptides for expression of multiple genes in Dictyostelium discoideum."

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Data are organized into separate folders for each figure within 0_DATA; the list below references separately-uploaded Python code for analysis and plotting, which may be run to reproduce the plots and figures using the environment specified in 2A_environment.yml in 1_SOFTWARE. To run this code, the 0_DATA and 1_SOFTWARE folders should share a parent folder and the code should be run from inside the 1_SOFTWARE folder. 

See linked publication for additional details. 

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Figure 1: ‘Fig 1 cleavage’
Original TIFs for microscopy images are in ‘Microscopy’, along with corresponding JPEGs used to make figures. 
Each file name includes the construct label (flex = flexible linker, or P2A, T2A, E2A, F2A), and channel (DIC, RFP, GFP). Merge (jpg only) = composite of RFP and GFP channels. TIF file names also include laser power (laser50 = 50%), exposure time in milliseconds (exp200 = 200 ms). 
Files with the subscript _crop.tif are multi-channel tif files containing DIC, RFP and GFP. 
The single-channel tifs were extracted from these files. 
The file metadata.csv includes descriptions of each tif file, including which linker was used. 

Original mCherry western blot files are in ‘Western Blots’. 
Three replicates are loaded for each mCherry-H2B-2A-mNeonGreen construct in the order: linker, P2A, T2A, F2A, E2A.
The original data is provided in western_quantification.xlsx. In this file, Sheet 1 contains the values quantified from the regions of interest. Name = ROI label. Condition = construct, Replicate = experimental replicate, Band = which band (cleaved or uncleaved), Vol. (Int.) = total intensity throughout the volume in arbitrary units. Local Bg. Corr. Vol. = total intensity after background subtraction (arbitrary units). Median Intensity = median background-corrected value (arbitrary units). Area = size of the region of interest (pixels). Sheet 2 contains the same values organized by linker construct and replicate, along with the calculated percent cleaved for each replicate. Sheet 3 contains the average percent cleaved for each treatment condition. 

The western_tif subfolder contains the original image files for each channel (raw data, saved as .tif). The western_png subfolder contains .png files for the membrane, total protein, and chemiluminescence channels alone and composite images for total protein and chemiluminescence with the membrane; these images were rotated by 3 degrees to align the bands horizontally using the iBright software. Membrane = membrane imaging mode to capture visible colorimetric staining; No-Stain Labeled Membrane = fluorescence image of the membrane labeled with No-Stain protein labeling reagent from Invitrogen to show total protein amounts in each lane; SuperSignal West Pico Plus = chemiluminescence image of the membrane incubated with Recombinant Anti-mCherry antibody [EPR20579] and HRP-conjugated Goat anti-Rabbit IgG, Invitrogen G21234. 

The western blot image used for display in the figure is 2022_08_08_Fig1Brevised.png, and representative microscopy data and diagrams of the constructs are shown in 2021_10_19_Fig1A_gm_DIC_bw.png.

Running ‘fig1.py’ generates Figure 1 using these files. 

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Figures 2 and 3: ‘Fig 2-3 Order Effects’
Original flow cytometry data is in subfolders.
Data is for AX4 D. discoideum cells transfected with mNeonGreen and mScarlet-I linked by a flexible linker, P2A, T2A, or expressed as separate transcriptional units on the same plasmid, with either mNeonGreen or mScarlet-I first. 
Running ‘plotallreplicates.py’ generates supplementary figures S3-S10
Subsequently running ‘fig_ordereffectsandtranscriptlength.py’ generates Figures 2-3
The file that ends with labels.png shows the constructs used in this experiment. 

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Figure 4: ‘Fig 4 DC vs P2A hygro’
Original flow cytometry data is in subfolders.
Data is for AX4 or NC28.1 D. discoideum cells transfected with mNeonGreen and hygromycin resistance either linked by P2A or as separate transcriptional units on the same plasmid, grown with different concentrations of Hygromycin B Gold (Invitrogen). File names are of the format: {expt identifier}_{strain}_{construct}_{concentration}.fcs. In this format, strain identifies the D. discoideum strain used (AX4 or NIB=NC28.1); construct indicates the transfection plasmid (DC = dual transcriptional unit, P2A = P2A linker, noDNA = no DNA control); and concentration indicates the concentration of hygromycin used in micrograms/mL. 
Running ‘DCvsP2A_supp.py’ generates supplementary figures S12-S23
Subsequently running ‘DCvsP2A_mainfig.py’ generates Figures 4
The file that ends with labels.png shows the constructs used in this experiment. 

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Figure S1: ‘S1_mNG_alone’
Original TIFs for microscopy images of AX4 D. discoideum cells transfected with mNeonGreen are in ‘Microscopy’. Scale for these images: 7.6923 pixels per micron. 
Running ‘generateFigS1_mNG.py’ generates Figure S1

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Figure S2: ‘S2 Gating fig’
The .fcs files include the flow cytometry data used to design gating. 
-Group_AX4_mCherry.fcs: D. discoideum AX4 transfected with mCherry (red only)
-Group_1_23.1_mNG.fcs: D. discoideum AX4 transfected with mNeonGreen (green only)
-Group_2_24.1_mS.fcs: D. discoideum AX4 transfected with mScarlet-I (red only)
-Group_3_35.6_mS-T2A-mNG.fcs: D. discoideum AX4 transfected with mScarlet-I-T2A-mNeonGreen (red and green construct)
-Group_4_noDNA.fcs: D. discoideum AX4 after mock transfection with no DNA (negative selection control; dead cell control)
-Group_5_Klebs.fcs: Klebsiella aerogenes (bacterial food source)
-Group_6_AX4_untransfected_fromplate.fcs: D. discoideum AX4 with no transfection and no selection (non-fluorescent live cells)

Running ‘supplfiggatingandcorrectioncurve.py’ generates Figure S2

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Figure S11: ‘S11 Antibiotic’
Cells were grown with the indicated concentration of Hygromycin B Gold (Invitrogen), and seeded in a 5-fold dilution series across the columns of the plate.
The csv file contains the maximum dilution at which fruiting bodies were observed within 2 weeks (number of wells with fruiting bodies) for each condition. 
Running ‘hygrotest.py’ generates Figure S11 B and C

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