Detection of protein - synthesizing microorganisms in the environment via bioorthogonal non - canonical amino acid tagging ( BONCAT )
Directly add AHA or HPG using a sterile - filtered ( 0.2 μM ) , pH - adjusted ( pH 7.0 ) stock solution yielding a final concentration of 1 nM to 1 mM .
Additionally , perform replicate experiments and include replicated incubations without AHA / HPG .
For L - 2 - amino - 4 - azidobutanoic acid ( L - azidohomoalanine , AHA ) : Dissolve in nano - pure water , adjust to pH 7.0 , filter sterilize ( 0.2 μm ) , and store in the dark at 4 C .
Prepare stock solutions of 1–100 mM .
For L - 2 - amino - 5 - hexynoic acid ( L - homopropargylglycine , HPG ) : Dissolve in nano - pure water , adjust to pH 7.0 , filter sterilize ( 0.2 μm ) , and store in the dark at 4 C .
Prepare stock solutions of 1–100 mM .
Fix cells according to standard protocols [ 34 ] immediately after sampling either by ( 1 ) fixation in 3 % formaldehyde ( PFA ) in PBS ( See Steps 3 - 6 ) or ( 2 ) by resuspending pelleted biomass in a 1 : 1 mix of PBS : EtOH .
( See Step 7 ) For fixation with PFA , pellet the biomass , remove the supernatant ( SN ) , and resuspend cells in 3 % PFA in PBS .
For aqueous samples , directly add PFA to reach a final concentration of 3 % PFA .
Fix for either 3 h on ice or 1 h at RT .
After fixation , Pellet the biomass by centrifugation or filter onto 0.2 μm filters .
Wash with PBS to remove remaining PFA before resuspending biomass in 1 : 1 PBS : EtOH .
Store at - 20°C .
Make sure to deposit PFA in the chemical waste .
For EtOH fixation , pellet biomass , remove supernatant , resuspend in 1 : 1 PBS : EtOH , and store at - 20°C .
Immobilize biomass either on glass slides or filters .
Dry at 46°C or , if not available , at 37°C or RT .
Dehydrate and permeabilize cells by sequentially placing slides or filters for 3 min into 50 mL tubes that contain 50 , 80 , and 96 % ethanol .
Dry biomass using pressurized air .
Pellet sample via centrifugation ( 16,100g or max .
setting for 5 min at RT ) and resuspend in 250 μL 80 % EtOH .
Mix by vortex and incubate for 3 min at RT .
Add 1.5 mL 96 % EtOH , mix by vortex , and incubate for 3 min at RT .
Afterwards , pellet sample via centrifugation and resuspend in 221 μL PBS .
Removing small volumes of leftover EtOH is not necessary as it does not interfere with the click reaction .
If using immobilized biomass , after dehydration of the sample , prepare the dye premix by mixing 1.25μL of 20 mM CuSO4 solution with 2.50μL of 50 mM THPTA and 0.30μL of alkyne dye .
Allow to react for 3 min at RT in the dark .
We recommend to perform Cu ( I ) - catalyzed click chemistry at a dye concentration of 1 - 5μM ( final concentration ) to guarantee for best signal - to - noise ratios , but substantially lower or higher concentrations can be used , if necessary .
We successfully tested concentrations as low as 10 nM and as high as 50μM .
If using immobilized biomass after dehydration , continue .
Otherwise , skip to Step 18 .
Add 12.5 μL of each 100 mM sodium ascorbate and 100 mM aminoguanidine hydrochloride to 221 μL PBS .
Then , add the dye premix and invert the tube once ( do not mix by vortex to maintain reducing conditions ) .
Cover the sample with 20 μL of the click solution , transfer the slide into a humid chamber ( water on tissue paper ) , and incubate in the dark at RT for 30 min .
Increasing the incubation time is possible , but typically does not increase fluorescence signal .
Wash the slide or filter three times for 3 min each in PBS - filled 50 mL tubes before dehydrating it by incubating it for 3 min in 50 % EtOH at RT .
If the biomass is in solution , all reagents ( sodium ascorbate and aminoguanidine , followed after 3 min by the dye premix , final concentrations as described above ) are added directly to the sample .
Invert tubes once and incubate in the dark at RT for 30 min .
Afterwards , wash samples three times with PBS and then one time in 50 % EtOH ( RT ) .
Between washing steps , pellet samples via centrifugation for 5 min at 16,100g ( or highest setting ) at RT .
Finally , resuspend biomass in a 1 : 1 mix of PBS : EtOH , transfer onto a glass slide , and air - dry
