TITLE: BONCAT-FACS on river water and sewage effluent samples
AUTHORS: Katharine Moss, Tim Goodall, Daniel S Read

[DESCRIPTION]
Bioorthogonal non-canonical amino acid tagging (BONCAT) is a method for detecting translational activity at the single cell level. Briefly, samples are incubated with a non-canonical amino acid which gets incorporated into newly synthesised proteins. Samples are fixed, then reagents are added to fluorescently tag the non-canonical amino acid in new proteins.
BONCAT can then be combined with fluorescent activated-cell sorting (FACS) to sort the BONCAT active population from the total community. The sorted BONCAT active population can undergo DNA extraction for 16S rRNA sequencing.
This protocol is for river water and sewage effluent samples. 

This protocol was developed by the UKCEH Molecular Ecology group. 


[GUIDELINES]
Wear nitrile gloves and lab coat.

[STEPS]
SECTION: Advance reagent preparation
1. Prepare 100 mg/mL  HPG solution by dissolving 100 mg  of HPG in 1 mL  of MilliQ water. 

Store solution at 4 °C in the dark.
SECTION: Advance reagent preparation
2. To prepare 20 millimolar (mM)  CuSO4. 5H2O solution, dissolve 0.5 g of CuSO4. 5H2O in 100 mL  of MilliQ water.

Store solution at 4 °C .
SECTION: Advance reagent preparation
3. To prepare 1.15 Molarity (M)  THPTA stock solution, dissolve 500 mg  of THPTA in 1 mL  of MilliQ water.

To prepare 50 millimolar (mM)  THPTA working solution, dilute 10 µL  of stock solution in 220 µL  of MilliQ water.

Store solutions at -20 °C .
 

SECTION: Advance reagent preparation
4. To prepare 6.5 millimolar (mM)  Alexa Fluor 488 picolyl azide stock solution, dissolve 5 mg  of Alexa Fluor 488 picolyl azide in 1 mL  of molecular grade DMSO.

To prepare 0.13 millimolar (mM)  Alexa Fluor 488 picolyl azide working solution, dilute 10 µL  of stock solution in 490 µL  of molecular-grade DMSO.

Store solutions at -20 °C  in the dark.

 

SECTION: Reagent preparation on the day
6. Make fresh 100 millimolar (mM)  sodium ascorbate solution.

To prepare, dissolve 0.8 g  of sodium ascorbate in 40 mL  of 1X PBS.

SECTION: Advance reagent preparation
5. Prepare 50 % (v/v) , 80 % (v/v)  and 96 % (v/v)  ethanol solutions with MilliQ water.

SECTION: Reagent preparation on the day
7. Make fresh 100 millimolar (mM)  aminoguanidine hydrochloride solution.

To prepare, dissolve 0.4 g  of aminoguanidine hydrochloride in 40 mL  of 1X PBS.
SECTION: Non-canonical amino acid incorporation
9. To 25 mL  of river water, add 32 µL  of  100 mg/mL  HPG. Final concentration of HPG in sample is 1 millimolar (mM) . 

Incubate in the dark at Room temperature for 15 min .

 

 

SECTION: Non-canonical amino acid incorporation
10. Fix sample with 25 mL  of molecular-grade ethanol (final concentration 50 % (v/v) ). 

Incubate in the dark at Room temperature  for 60 min .

SECTION: Reagent preparation on the day
8. Before use, remove all reagents from fridge/ freezer and allow to come up to/ thaw at Room temperature .

SECTION: Biomass filtration and dehydration
11. Set up filter funnels (25 mm diameter) with PVDF filters (25 mm, 0.2 μm).  Connect to vacuum pump or tap.

Label funnels with sample names.
SECTION: Biomass filtration and dehydration
12. Condition filters with approximately 5 mL  of 50 % (v/v)  ethanol.

SECTION: Biomass filtration and dehydration
13. Filter the ethanol fixed samples onto the filters.
SECTION: Biomass filtration and dehydration
14. Release vacuum and cover filters with 0.5 mL  of 80 % (v/v)  ethanol.

Incubate for 3 min , then vacuum through the ethanol.

SECTION: Biomass filtration and dehydration
15. Release vacuum and cover filters with 0.5 mL  of 96 % (v/v)  ethanol.

Incubate for 3 min , then vacuum through the ethanol.

Vacuum dry the filter for approximately 10 min .

SECTION: Cu catalysed azide-alkyne cycloaddtion (click reaction)
16. The volumetric amounts of reagents in steps 16 and 17 are for one sample's worth. It is recommended to make enough for n + 1 samples (n = number of samples).

To make dye premix, add together 2.5 µL  of CuSO4, 5 µL  of THPTA and 19 µL  of Alexa Fluor 488 picolyl azide. Invert tube.

Incubate in the dark at Room temperature  for 3 min .

SECTION: Cu catalysed azide-alkyne cycloaddtion (click reaction)
17. To dye premix, add 25 µL  of sodium ascorbate, 25 µL  of aminoguanidine hydrochloride, and 423.5 µL  of 1X PBS. Invert tube.

Final concentration of Alexa Fluor picolyl azide in reaction mixture is 5 micromolar (µM) .

 


 


SECTION: Cu catalysed azide-alkyne cycloaddtion (click reaction)
18. Release vacuum on filter funnels, and cover filters with 0.5 mL  of reaction mixture.

Incubate in the dark (e.g., cover funnels with foil) at Room temperature  for 30 min .

SECTION: Filter washes
19. Vacuum through reaction mixture.
SECTION: Filter washes
20. Release vacuum and cover filter with 0.5 mL  of 1X PBS.

Incubate for 3 min , then vacuum through.

Repeat twice more.
SECTION: Filter washes
21. Repeat step 20 once with 50 % (v/v)  ethanol.

SECTION: Filter washes
22. Vacuum dry filters for approximately 10 min .

SECTION: Resuspension of cells
23. With clean tweezers, place filters into 2 ml o-ring tubes with the top of the filter facing inwards.
 

SECTION: Resuspension of cells
24. Add six clean stainless steel ball bearings to each tube.
 

SECTION: Resuspension of cells
25. Add 1.5 mL  of 1X PBS to each tube.

SECTION: Resuspension of cells
26. Vortex/shake tubes at max speed for 3 min .

SECTION: Sample storage
27. Aliquot 1 mL  of bacterial suspension into 2 ml o-ring tubes.

SECTION: Sample storage
28. Add 1 mL  of molecular-grade ethanol to suspension. Final ethanol concentration is 50 % (v/v) .

Can be stored at -20 °C  for up to 2 weeks.

SECTION: Fluorescent activated-cell sorting and DNA extraction
29. Dilute 300 µL of ethanol-fixed sample in 200 µL of sterile-filtered 1X PBS

SECTION: Fluorescent activated-cell sorting and DNA extraction
31. Run HPG-tagged samples according to step 30. Sort 500,000 events from the BONCAT positive gate in semi-purity mode. Collect sorted events in 5 ml flow cytometry containing tubes containing 1 mL of sterile-filtered 1X PBS.
 
.

SECTION: Fluorescent activated-cell sorting and DNA extraction
30. Run a negative control to determine the BONCAT positive gate on a SH800S cell sorter at sample pressure of 5. Draw the BONCAT positive gate to encompass less than 0.1% of the negative control population on a AF 488 vs. FSC density plot. Use a 488 nm laser for excitation, and set threshold to 2 on FSC.
SECTION: Fluorescent activated-cell sorting and DNA extraction
32. Vacuum concentrate sorted populations to dryness using a vacuum concentrator.
SECTION: Fluorescent activated-cell sorting and DNA extraction
33. Store pellets at -80 °C until DNA extraction.

SECTION: Fluorescent activated-cell sorting and DNA extraction
34. For DNA extraction, follow the manufacturer's standard protocol for the Qiagen DNeasy UltraClean Microbial kit.