Updated figure and added protocols

README.md

@@ -28,7 +28,6 @@ measurements.
 * `visualization`: This directory houses Python scripts used in generating all 
 of the figures in this work. 
 
-
 ## `data`
 This is where all of the (small) datasets used in this work live. It is broken 
 down in to groups of literature data and our own measurements. Further documentation 
@@ -40,6 +39,10 @@ This directory houses all of the figures used in the manuscript, usually separat
 by figure panel. As of now, this houses many different versions used in different 
 stages of the manuscript.
 
+## `protocols`
+This directory houses markdown files with media recipes and detailed step-by-step
+guides for the various experimental protocols performed in this work.
+
 ## `software`
 This project required the development of an independent software package, primarily 
 for the image processing. This module (named `size`) is packaged and documented

code/processing/mass_spectrometry/ms_processing.py

@@ -1,4 +1,5 @@
 # %%
+
 import numpy as np
 import pandas as pd
 data = pd.read_csv(
@@ -7,11 +8,10 @@
 data['cyto_enrichment'] = data['cyto_intensity'] / data['proteome_intensity']
 data['peri_enrichment'] = data['peri_intensity'] / data['proteome_intensity']
 data['relative_signal'] = data['peri_intensity'] / data['cyto_intensity']
+data['fractional_peri'] = data['peri_intensity'] / data['proteome_intensity'].sum()
+data['fractional_cyto'] = data['cyto_intensity'] / data['proteome_intensity'].sum()
 data = data[['gene', 'localization', 'cyto_enrichment',
-             'peri_enrichment', 'relative_signal']]
+             'peri_enrichment', 'relative_signal', 'fractional_peri', 'fractional_cyto']]
 data.dropna(inplace=True)
 data.to_csv(
-    '../../../data/mass_spectrometry/periplasm_fractionation_confirmation.csv', index=False)
-# melted = data.melt(id_vars=['gene', 'localization'])
-# melted.dropna(inplace=True)
-# melted.rename()
+    '../../../data/mass_spectrometry/periplasm_fractionation_confirmation.csv', index=False)

code/visualization/FigAX_peri_conf.py

@@ -0,0 +1,16 @@
+#%%
+
+import numpy as np 
+import pandas as pd 
+import matplotlib.pyplot as plt
+import size.viz
+cor, pal = size.viz.matplotlib_style()
+ms_conf = pd.read_csv(
+    '../../data/mass_spectrometry/periplasm_fractionation_confirmation.csv')
+
+grouped = ms_conf[ms_conf['localization']=='periplasm'][['fractional_peri', 'fractional_cyto']].sum().reset_index()
+grouped
+
+fig, ax = plt.subplots(1,1, figsize=(1, 1))
+ax.bar(['supernatant', 'pellet'], grouped[0].values)
+ax.set_ylabel('fraction of total intensity', fontsize=6)

code/visualization/FigS3_inner_outer_membrane_densities.py

@@ -17,23 +17,23 @@
 ax[0, 1].set_ylim([0, 7])
 ax[1, 1].set_ylim([0, 7])
 for g, d in ms_data[ms_data['localization'] == 'inner membrane'].groupby('dataset_name'):
-    fmt = size.viz.style_point(g)
+    fmt = size.viz.style_point(g, alpha=0.45)
     fmt['color'] = cor['pale_blue']
     ax[0, 0].plot(d['growth_rate_hr'], d['mass_frac'], **fmt, ms=4)
 
 for g, d in ms_data[ms_data['localization'] == 'outer membrane'].groupby('dataset_name'):
-    fmt = size.viz.style_point(g, alpha=0.75)
+    fmt = size.viz.style_point(g, alpha=0.45)
     fmt['color'] = cor['blue']
     ax[1, 0].plot(d['growth_rate_hr'], d['mass_frac'], **fmt, ms=4)
 
 axes = {'ms_rho_mem_inner': [ax[0, 1], cor['pale_blue']],
         'ms_rho_mem_outer': [ax[1, 1], cor['dark_blue']]}
 for g, d in densities.groupby(['quantity', 'source']):
-    fmt = size.viz.style_point(g[1], alpha=0.75)
+    fmt = size.viz.style_point(g[1], alpha=0.45)
     _axes = axes[g[0]]
     fmt['color'] = _axes[1]
     _axes[0].vlines(d['growth_rate_hr'], d['2.5%'],
-                    d['97.5%'], lw=0.5, color=fmt['color'])
+                    d['97.5%'], lw=0.5, color=fmt['color'], alpha=0.45)
     _axes[0].plot(d['growth_rate_hr'], d['median_value'], **fmt, ms=4)
 
 for a in ax.ravel():

protocols/N-C-_medium_recipe.md

@@ -0,0 +1,32 @@
+# N-C- Base Minimal Medium 
+
+## Brief Description
+
+N-C- Minimal Medium is effectively a phosphate buffered base medium for growth of heterotrophic microbes. It is typically stored as a 10X stock to which carbon and nitrogen sources are later added to make a complete medium capable of supporting microbial growth. It is a high-osmolarity medium, so care should be taken when adding in large amounts of Carbon and Nitrogen sources.
+
+## Composition
+
+| Component | Concentration [mM] | Function | Vendor & Catalog Number |
+| --- | --- | --- | --- |
+| $K_2SO_4$ | 5.7 | Sulfate Source | Sigma-Aldrich: P0772 |
+| $K_2HPO_4$ | 77.6 | Buffering + phosphate source | Sigma-Aldrich: P3786 |
+| $KH_2PO_4$ | 34.6 | Buffering + phosphate source | Sigma-Aldrich: P5655 |
+| $MgSO_4$ | 0.4 | Magnesium + sulfate source | Sigma-Aldrich: M2643 |
+| $NaCl$ | 43.1 | Sodium source + osmolarity control | Sigma-Aldrich: S3014 |
+| Carbon Source | Variable | Carbon source | Variable |
+| $NH_4Cl$ | 10 | Nitrogen Source | Sigma-Aldrich: A9434 |
+
+## Recipe
+
+It’s recommended to store the N-C- buffer base as a filter-sterilized 4x stock. This has a long shelf life (>  2 years) and is robust to contamination as it doesn’t have either a nitrogen or carbon source.
+
+### **500 mL of  a 4X N-C- Base Buffer**
+
+| Component | Quantity | Note |
+| --- | --- | --- |
+| $K_2SO_4$ | 2 g |  |
+| $K_2HPO_4$ | 27 g | Can also use 35.4 g of $K_2HPO_4\cdot 3H_2O$ |
+| $KH_2PO_4$ | 9.4 g |  |
+| $MgSO_4$ | 0.8 mL of 1M stock | Can also add 0.2 g of $MgSO_4\cdot 7H_2O$ |
+| $NaCl$ | 5g |  |
+| dd $H_2O$ | to 500 mL |  |

protocols/README.md

@@ -0,0 +1,4 @@
+#`protocols`
+
+Here lie markdown files with medium recipes and experimental protocols as defined
+in the paper and appendix.

protocols/membrane_protein_quantification_protocol.md

@@ -0,0 +1,57 @@
+
+# Overview
+
+Protocol developed by Roshali de Silva and Jonas. Last edits summer 2023. 
+
+# Required reagents
+- PBS buffer: pH 7.4 (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 24.2 mM, KH2PO4 5.2 mM)
+- Spheroplast buffer (0.1 M Tris pH 8.0, 500 mM sucrose, 0.5 mM EDTA pH 8.0)
+- 100 mM MgCl2
+- 2% SDS
+- BCA protein assay (Thermo Fisher Micro BCA Protein Assay Kit, 23235)
+
+# Centrifugation settings
+- Standard centrifugation: 13k RPM for 1.5 min.
+- Ultracentrifugation: 65k RPM. 
+
+# Equipment and consumables
+- Ultracentrifuge: Beckman Coulter OptimaMAX Ultracentrifuge for up to 130k RPM.
+- Regular centrifuge for microtubes.
+- Rotor: TLA-120.1. Rotor can handle 14 tubes (seven conditions) at a time and supports the min of 100k G required. Rotor storage at 4C. 
+- 0.5 ml ultracentrifugation tubes (Beckman Coulter tube 343775, 8x34mm).
+- Plate reader for absorbance measurements: BioTek Epoch2
+- 96-well plates (e.g. Greiner CELLSTAR 655 185) and 2ml microtubes.
+
+# Part 1 - Sample taking
+1. Collect cell samples from steadily growing cultures (2x 1ml in 1.5 ml Eppendorf tube, OD600~0.5, standard centrifugation and remove supernatant)
+2. For LB cultures only, resuspend the pellet in 1ml of PBS. Obtain pellet (standard centrifugation and remove supernatant)
+3. Freeze samples at -80. Freeze at least for 30 minutes but samples can also be stored overnight or longer. Note: Freezing is required and cannot be skipped.
+
+# Part 2 - Cell lysis
+4.	Thaw samples at room temperature for ~5 min. 
+5.	Add 500ul cold spheroplast buffer and resuspend the pellet. 
+6.	Incubate on ice for 20 min.
+7.	Add 500ul 100 mM MgCl2 and mix by pipetting. Incubate on ice for 10 min.
+8.	Quickly spin down tubes to collect liquid. Transfer liquid to a 15ml Falcon tube to prepare for sonication (keep Eppendorf tube). Make sure the liquid is exclusively at the bottom of the tube.
+9.	Keep falcon tubes on ice. Transfer to sonication machine and run sonication. Making sure sonication dip has been deeply emerged in Falcon tube. 3x 30-second pulses with 5 sec breaks in between. 
+10.	Take total protein samples (optional). Collect 20ul of supernatant and add to a new tube. Add 280 ul 2% SDS and mix by pipetting. Heat at 100C for 30 min. Centrifuge (standard setting), no pellet should be visible. Store sample for further protein quantification (total protein sample)
+11.	Transfer the liquid from the Falcon tube back to the 1.5 ml Eppendorf tube.
+12.	Centrifuge (Standard settings), about 1ml supernatant.
+13.	Transfer 500ul of supernatant into an ultracentrifugation tube. Transfer the remaining supernatant (~450ul) to another ultracentrifugation tube. 
+14.	Quantify proteins of residual pellet. The remaining pellet contains unlysed cells. To quantify the protein content of those, resuspend the pellet in 500ul of 2% SDS. Heat at 100C for 30 min. Centrifuge (standard setting), no pellet should be visible. Store the sample for further protein quantification (residual pellet sample).
+# Part 3 - Run Ultracentrifugation
+15.	Balance tube pairs by measuring all tubes to obtain a match in weight (less than 0.01g difference between tubes). To match, add spheroplast buffer to lighter tubes.
+16.	Arrange balanced tubes on opposite site of rotor. Ultra-centrifuge for 65k RPM for 1h at 4-7C. 
+17.	Take tubes. Pellets might be invisible. Completely remove the supernatant and transfer to new Eppendorf tube. Best to use a P200 pipette and remove supernutant sequentially in 3 steps. When removing the final volume, put the tip straight down to the bottom (do not touch sides as pellet is located there). Store the supernatant which contains cytoplasmic and periplasmic fraction (cytoplasmic sample)
+Digest membrane proteins
+18.	Add 150ul 2% SDS and resuspend (invisible) pellet thoroughly by pipetting (30-60 seconds per sample). Do this after centrifugation without adding a longer break to prevent pellets from drying out. Store the samples which contain the membrane fraction (membrane sample).
+# Part 4 - BCA assay for protein quantification
+19.	Preparation: Mix all three assay reagents according to the manual in a ratio of 25:24:1 to obtain final working reagent. Per sample, 150 ul of final working reagent is needed.
+20.	Preparation: Turn on plate reader and set incubation to 37C. 
+21.	Transfer samples to wells of 96-well microtiter plate. For membrane samples use 150ul undiluted samples. For cytoplasmic/total/residual pellet samples dilute samples 15x and use 150ul of diluted sample. Note: membrane samples contain a high SDS content. Minimize bubble formation by minimizing pipetting.
+22.	After all samples have been transferred to plate, add 150ul of working reagent to each well. 
+23.	Transfer plate to plate reader and start protocol:
+-	Incubation at 37C
+-	Start with 30 second shaking period
+-	Incubate plate for 2h (no shaking)
+-	Take absorbance readings at 562. 

protocols/periplasm_quantification_protocol.md

@@ -0,0 +1,32 @@
+
+# Overview
+
+Protocol developed by Roshali and Jonas. Last edits Summer 2023.
+
+# Required reagents
+- PBS buffer: pH 7.4 ( 137 mM NaCl, 2.7 mM KCl, 24.2 mM Na2HPO4, 5.2 mM KH2PO4)
+- Spheroplast buffer (0.1 M Tris pH 8.0, 500 mM sucrose, 0.5 mM EDTA pH 8.0)
+- 1mM MgCl2 solution
+- Bio-Rad protein assay (catalogue number 5000002)
+
+# Standard settings centrifugation
+- Temperature of 4C, 8000 RPM for 8 min.
+
+# Equipment and consumables
+- Regular centrifuge for microtubes.
+
+# Part 1 - Separation periplasmic and other proteins.
+1. Collect cell samples by transferring 1ml to 2ml microbe (culture OD600~0.5).
+2. Centrifuge and remove supernatant
+3. Resuspend cell pellet in 400 ul PBS at pH 7.4, centrifuge, and remove supernatant.
+4. Resuspend cell pellet in 400 ul spheroblast buffer and incubate tube for 5 min on ice. 
+5. Centrifuge sample and carefully remove supernatant. 
+6. Resuspend cell petter in 400 ul of hypotonic 1mM MgCl2 solution and incube for 2 minutes on ice. 
+7. Transfer the supernatant to a new microbe. Optionally, store the remaining pellet contining mostly cytoplasmic proteins at -80C for further protein quantification via the biuret assay. 
+
+# Part 2 - Protein quantification with the Bio-Rad protein assay kit. 
+8. Mix 800 ul of protein sample with 200 ul the Bradford dye solution. 
+9. Measure the absorbance at a wavelength of 595 nm. 
+
+# Standard curve
+Standard curves were generated using BSA and IgG over the concentration ranges mentioned in the manual. 

protocols/total_RNA_protocol.md

@@ -0,0 +1,37 @@
+
+# Overview
+
+Version of the classical perchloric acid assay. Protocol notes added by Richa and Jonas, last edits Summer 2023.
+
+# Required reagents
+- 0.7M HClO4
+- 3M HClO4
+- 0.5M HClO4
+- 0.3M KOH
+Have all HClO4 stocks cold and keep on ice during the procedure. Stock solutions are stable at RT.
+
+# Standard settings centrifugation
+- Temperature of 4C, 8000 RPM for 8 min.
+
+# Equipment and consumables
+- Regular centrifuge for microtubes.
+- UV-Vis spectroscope (Thermo XY)
+
+# Protocol steps
+1. Take 1.5ml sample from culture and transfer to a 2ml microtube. 
+2. Centrifuge sample, remove supernatant, and put microtube on dry ice. Measure OD600 of supernatant to account for cell loss via supernatant. 
+3. Wash cells twice with 600 µl cold 0.7M HClO4 (suspension of pellet, centrifugation, subsequent supernatant removal and measurement OD600 of supernatant for cell loss correction). 
+4. Digest cells by adding 300 µl 0.3M KOH for 60 minutes at 37C. Mix from time to time. 
+5. Add 100ul 3M HClO4 to extract
+6. Centrifuge and collect supernatant into a new 2ml collection microtube. 
+7. Wash the remaining precipitate twice with 550ul cold 0.5M HClO4 and each time add the supernatant to the collection tube. 
+8. Centrifuge the 1.5 ml liquid in the collection tube to spin down any remaining precipitates.
+9. Read OD260 of the supernatant (typical range between 1 and 3) using 500ul liquid volume and quarts cuvette.
+10. Calculate the RNA amount as R(µg/ml/OD600) = OD260*31/OD600.
+
+# Notes
+The converting factor 31 is based on that the molar extinction coefficient is 10.5/mmol/cm and the average
+molecular weight of an E. coli RNA nucleotide residue is 324. Reported molar extinction coefficients ranges from 10 to 10.8 in the literature.
+
+# Reference
+Benthin, Nielsen, and Villadsen. A simple and reliable method for the determination of cellular RNA content. Biotechnology Techniques 5 39-41, 1991.

protocols/total_protein_protocol.md

@@ -0,0 +1,37 @@
+
+# Overview
+
+The buiret method for total protein quantification. Protocol notes added by Richa and Jonas, last edits Summer 2023.
+
+# Required reagents
+- 3M NaOH
+- 1.6% w/v CuSO4
+- 10mg/ml BSA stock. Store BSA stock at 4C
+
+# Standard settings centrifugation
+- Temperature of 4C, 8000 RPM for 8 min.
+
+# Equipment and consumables
+- Regular centrifuge for microtubes. Standard setting: 13k, 1 min
+- Vis spectroscope (Thermo XY)
+
+# Preparation
+- Turn heat block on 100C.
+- For standard curve prepared in 1.5 ml tubes reagent blanks containing 200 ul of water and 200 ul of different BSA stock dilutions.
+
+# Part 1 - Washing
+1. Collect 1.8 ml of cell culture (OD600 > ~ 0.5) into a 2 ml tube. Centrifuge. Pipetting as much supernatant as possible to the cuvette without disrupting the pellet and read OD600 for cell loss correction.
+2. Wash cell pellets with 1 ml of NaCl solution with similar osmolality as the cell suspension. After centrifugation, pipette from the tube as much supernatant as possible into cuvette and read OD600 for cell loss correction.
+3. Resuspend cell pellets in 200 ul of water. Transfer 200 ul of the cell suspension to a new microtube and place it on dry ice.
+4. Add 1ml of water to the old tube and measure OD600 for cell loss correction. 
+
+# Part 2 - Extraction and measurement
+5. Add 100ul 3M NaOH to each tube. Place tubes on the heat block for 5 min and cool for an additional 5 min at RT.
+6. Add 100ul 1.6% CuSO4 to each tube, shake thoroughly and let tubes sit for 5 min. 
+7. Centrifuge the tubes at max speed for 3 min. Read OD555. 
+
+# Notes
+The concentration of the BSA stock can be verified by measuring its OD280. The BSA concentration in unit of mg/ml is equal to OD280/0.63  (http://www.ruf.rice.edu/~bioslabs/methods/protein/abs280.html). 
+
+# Reference 
+Herbert D., Phipps P. J., and Strange R. E.. Chemical analysis of microbial  cells, in Methods in Microbiology (edited by Norris J. R., & Ribbons D. W.),  1971, Academic Press.