May 20, 2025
Estimating the Relative Rates of Lipopolysaccharide Synthesis in Escherichia coli K-12 by Click Chemistry-Mediated Labeling
- Sheng Shu1,2,
- Wei Mi1,2
- 1Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA;
- 2Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA
Protocol Citation: Sheng Shu, Wei Mi 2025. Estimating the Relative Rates of Lipopolysaccharide Synthesis in Escherichia coli K-12 by Click Chemistry-Mediated Labeling. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld5bzov5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 13, 2025
Last Modified: May 20, 2025
Protocol Integer ID: 124535
Keywords: Lipopolysaccharide, LPS, Click-chemistry, Kdo, in-gel fluorescence, E. coli
Funders Acknowledgements:
Wei Mi
Grant ID: R01GM137068
Wei Mi
Grant ID: RM1GM149406
Abstract
Lipopolysaccharide (LPS), a critical glycolipid component of Gram-negative bacteria, plays a central role in bacterial membrane integrity and host immune interactions. Despite extensive studies on the regulation of LPS synthesis, methods to quantify its synthesis rate remain limited. Here, we present a novel approach to measure in vivo LPS synthesis rates in E. coli K-12 strain MG1655 using click chemistry. This method involves the incorporation of an exogenous Kdo analog, 8-azido-3,8-dideoxy-D-manno-oct-2-ulosonic acid (Kdo-N3), into newly synthesized LPS, followed by a copper-free click reaction with a fluorescent alkyne (AZDye 488 DBCO). The labeled LPS is separated by SDS-PAGE and visualized via in-gel fluorescence. We optimized the labeling conditions by testing different incubation times for Kdo-N3 and AZDye 488 DBCO, ultimately identifying a 10-minute Kdo-N3 incubation and 30-minute AZDye 488 DBCO labeling as optimal for quantifying LPS synthesis. Our results demonstrate that the amount of newly synthesized LPS correlates linearly with incubation time, particularly during the log phase of bacterial growth. This method offers a reliable, non-radioactive approach for measuring LPS synthesis in real-time, providing valuable insights into bacterial physiology and the regulation of LPS biogenesis.
Materials
Materials
- E. coli K-12 strain MG1655: The Coli Genetic Stock Center at Yale University
- Culture tube: Falcon, catalog number: 352059
- 250 ml flask: Pyrex, catalog number: 4980
- 15 ml centrifuge tube: Falcon, catalog number 352096
- 1.5 ml microcentrifuge tube: USA Scientific, catalog number: 1615-5500
- 0.2 µm syringe filter: Pall, catalog number: 4602
- Pro-Q Emerald 300 LPS staining kit: Thermo Fisher Scientific, Catalog number: P20495
- 15% SDS-PAGE gel: homemade
Recipe
M9-glucose minimal medium: dissolve 1.13 g M9 minimal medium salts (MP biomedicals, catalog number: 3037032) in 100 mL of Milli-Q water. After autoclave, add 200 µL of 1 Molarity (M) MgSO4 (Sigma, catalog number: 208094) (sterilized by autoclave), 10 µL of 1 Molarity (M) CaCl2 (J.T. Baker, catalog number: 1332-1) (sterilized by autoclave) and 1 mL of 40% glucose (Sigma, catalog number: G7021)(sterilized by filtering).
| A | B | |
| M9 minimal medium salts | 1.13 g | |
| Milli-Q water | 100 ml | |
| 1M MgSO4 | 200 µl | |
| 1M CaCl2 | 10 µl | |
| 40% glucose | 1 ml |
M9-maltose minimal medium: Dissolve 1.13 g M9 minimal medium salts (MP biomedicals, catalog number: 3037032) in 100 mL of Milli-Q water. After autoclave, add 200 µL of 1 Molarity (M) MgSO4 (Sigma, catalog number: 208094) (sterilized by autoclave), 10 µL of 1 Molarity (M) CaCl2 (J.T. Baker, catalog number: 1332-1) (sterilized by autoclave) and 1 mL of 20% maltose (Sigma, catalog number: M5895)(sterilized by filtering).
| A | B | |
| M9 minimal medium salts | 1.13 g | |
| Milli-Q water | 100 ml | |
| 1M MgSO4 | 200 µl | |
| 1M CaCl2 | 10 µl | |
| 20% maltose | 1 ml |
Kdo-N3 Stock solution (0.5 Molarity (M) ): Dissolve 100 mg of Kdo-N3 (Vector Laboratories, catalog number CCT-1241-100) in 714 µL of Milli-Q water. Filter the stock solution using a 0.2 µm syringe filter, aliquot to 20 µL in 1.5 ml microcentrifuge tubes, and store at -20 °C .
| A | B | |
| Kdo-N3 | 100 mg | |
| Milli-Q water | 714 µl |
AZDye 488 DBCO stock solution (10 millimolar (mM) ): Dissolve 5 mg of AZDye 488 DBCO (Vector Laboratories, catalog number CCT-1278) in 631 µL of DMSO. Aliquot to 20 µL in 1.5 ml microcentrifuge tubes, and store at -80 °C .
| A | B | |
| AZDye 488 DBCO | 5 mg | |
| DMSO | 631 µl |
Lysis buffer:
| A | B | |
| Tris-HCl, pH 7.8 | 50 mM | |
| Nacl | 300 mM | |
| Glycerol | 10 |
4×SDS loading buffer:
| A | B | |
| Tris-HCl (pH 6.8) | 0.25M | |
| SDS | 8% | |
| Glycerol | 40% | |
| Bromophenol blue | 0.02% |
SDS running buffer:
| A | B | |
| Tris base | 25 mM | |
| Glycine | 192 mM | |
| SDS | 0.10% |
Coomassie Blue stain solution:
| A | B | |
| Coomassie R-250 | 0.10% | |
| Methanol | 40% | |
| Acetic acid | 10% |
Coomassie Blue destain solution:
| Ethanol | 30% | |
| Acetic acid | 10% |
Equipment
1. Incubator shaker: any brand
2. Nanodrop spectrophotometer: Thermo Scientific, Catalog Number:13-400-525
3. Milli-Q water system: Millipore Milli-Q Reference A+
4. Microcentrifuge: Thermo Scientific, Sorvall Legend Micro 21
5. Centrifuge: Eppendorf model number 5810R
6. Sonication: BRANSON Sonifier 250
7. heating block: Benchmark BSH1002
8. Power source: Bio-Rad, PowerPac Basic
9. Gel running apparatus: Bio-Rad, Mini Protean Tetra Cell
10. Imager: Bio-Rad, ChemiDoc MP imager
11. Platform shaker: Benchmark, Orbi Shaker JR.
12. Scanner: Epson Perfection V600 Photo
Corning™ Falcon™Round-Bottom Polypropylene TubesFischer ScientificCatalog #352059
PYREX® 250 mL Narrow Mouth Erlenmeyer Flasks with Heavy Duty RimCorningCatalog #4980-250
15mLl polypropylene centrifuge tubesCorningCatalog #352096
Seal-Rite® 1.5 mL Microcentrifuge TubesUSA ScientificCatalog #1615-5500
Acrodisc Syringe Filters with Supor Membrane, Sterile - 0.2um, 13mm, 75/pkTisch ScientificCatalog #Pall 4602
Pro-Q™ Emerald 300 Lipopolysaccharide Gel Stain KitThermo FisherCatalog #P20495
M9 minimal medium saltsMP BiomedicalsCatalog #113037012-CF
Magnesium sulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #208094
JT Baker® 1332-01 Calcium Chloride, Dihydrate, Granular, BAKER ANALYZED™ ACS Reagent Grade, 500g PolCapitol ScientificCatalog #JTB-1332-01
D-( )-GlucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #G7021
D-(+)-Maltose monohydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M5895
Kdo AzideVector LaboratoriesCatalog #CCT-1241
AZDye 488 DBCOVector LaboratoriesCatalog #CCT-1278
Equipment
Nanodrop™ One Spectrophotometer with WiFi and Qubit™ 4 Fluorometer
NAME
Spectrophotometer
TYPE
Thermo Scientific™
BRAND
13-400-525
SKU
LINK
Kdo-N3 Incorporation into E. coli K-12 Cells
Kdo-N3 Incorporation into E. coli K-12 Cells
Inoculate a single colony of E. coli K-12 MG1655 strain into a culture tube containing 3 mL of M9-glucose medium. Grow the culture Overnight in a incubator shaker at 220 rpm, 37°C .
The following day, measure the optical density (OD) at 600 nm (OD600) using a Nanodrop spectrophotometer. Dilute the overnight culture into 50 mL of fresh M9-glucose medium in a 250 mL flask to achieve a starting OD600 of 0.05.
Allow the culture to grow in the incubator shaker at 220 rpm, 37°C until the OD600 reaches approximately 0.35 (log phase).
Transfer 2 mL of the log-phase culture to a new culture tube and add 4 µL of Kdo-N3 stock solution (0.5 Molarity (M) ). This will give a final Kdo-N3 concentration of 1 millimolar (mM) . Incubate the culture at in the shaker at 220 rpm, 37°C, 00:10:00 .
Click-chemistry to label Kdo-N3
Click-chemistry to label Kdo-N3
40m
40m
Transfer the samples to a 15 mL centrifuge tube and centrifuge at 2000 x g, Room temperature, 00:04:00 . Discard the supernatant and gently wash the cell pellets with 1 mL of M9-maltose medium by pipetting.
4m
Centrifuge the cells again at 2000 x g, Room temperature, 00:04:00 . Discard the supernatant and resuspend the cell pellet in 50 µL of M9-maltose medium.
4m
Transfer the resuspended cells to a sterile 1.5 mL microcentrifuge tube. Add 0.5 µL of AZDye 488 DBCO stock solution (10 millimolar (mM) ) to achieve a final concentration of 0.1 millimolar (mM) AZDye 488 DBCO. Wrap the microcentrifuge tube with aluminum foil and incubate it in a incubator shaker at 220 rpm, 37°C, 00:30:00 .
30m
After incubation, centrifuge the cells at 4500 x g, Room temperature, 00:02:00 using a microcentrifuge. Discard the supernatant and gently wash the cell pellet with 1 mL of M9-maltose medium by pipetting.
2m
Repeat step 8 to wash the cells two more times.
After the final wash, remove the wash medium and resuspend the cell pellets in 200 µL of lysis buffer.
SDS-PAGE and In-gel fluorescence
SDS-PAGE and In-gel fluorescence
30m
30m
Lyse the E. coli cells using a sonicator. After cell lysis, measure the absorbance at 600 nm using a Nanodrop spectrophotometer.
Nanodrop spectrophotometer to determine protein concentration.
Mix 30 µL of cell lysate with 10 µL of 4X SDS loading buffer. Boil the samples at 95 °C for 00:10:00 using a heating block.
10m
Load (50/ A280) µL of the prepared sample onto an SDS-PAGE gel. Run the gel at 180 V until the dye front reaches the bottom.
After electrophoresis, briefly rinse the gel with Milli-Q water. Capture an image of the gel using a Bio-Rad ChemiDoc MP imager.
Quantify the density of the LPS bands using ImageJ software. This will provide an estimate of the LPS synthesis rate.
Staining of the total LPS
Staining of the total LPS
After checking the in-gel fluorescence of AZDye 488, stain the gel for total LPS using the Pro-Q Emerald 300 LPS Staining Kit, following the manufacturer’s instructions.
Capture an image of the Pro-Q Emerald 300 fluorescence signal using the Bio-Rad ChemiDoc MP imager.
Staining of the total Protein
Staining of the total Protein
1h
1h
After visualizing the Pro-Q Emerald 300 signal for total LPS, stain the gel for total protein by incubating it in 50 mL of Coomassie Blue stain solution at Room temperature on a platform shaker set to 60 rpm, 00:20:00 .
20m
Discard the Coomassie Blue stain solution and rinse the gel twice with tap water.
Add 50 mL of Coomassie Blue destain solution and incubate the gel on a platform shaker at 60 rpm, 00:20:00 .
20m
Repeat Step 21 two to three times, or until the background is sufficiently clear.
Wash the gel with 50 mL of tap water for 00:20:00 to remove excess stain.
20m
Scan the gel using a scanner to capture the image.