HMA determination

Inoculate 5 ml of LB medium with 1% volume of E. coli culture and culture at 37℃ for 12h.200rpm

Prepare ZYM-5052 medium (5ml) by mix ingredients below: (for detailed recipe ZYM-5052.docx

A	B
ZY	4800μl
50×M	100μl
50×5052	100μl
1000×Trace element	5μl/10μl
1M MgSO4·7H2O	5μl/10μl
antibiotic	5μl
20% arabinose	50μl

Inoculate 5 ml of ZYM-5052 medium with 1% volume of E. coli LB culture and culture at 37℃ for 12h.200rpm

Prepare M9 medium (10ml) by mix ingredients below:

A	B
5 × M9	2ml
1M MgSO4·7H2O	20μl
1 M CaCl2	1μl
20% glucose	200μl
ddH2O	7.8ml

Centrifuge 6OD bacteria in 1.5 ml eppendorf (EP) tube at 4,200 x rpm for 10 minutes at room temperature.

Decant or aspirate and discard the culture media.

Use 200μl M9 medium to spirate and dis completely resuspend cell pellet and culture at 37℃ for 12h.200rpm

Centrifuge at 15,000 x g for 10 minute at room temperature.

Take 100 µl of supernatant into another EP tube and mix it with 900μl ddH2O.

Filter the sample with 0.22μm membrane filters and inject the sample into a clean sample bottle.

HMA was measured an HPLC (Agilent 1260) equipped with a UV detector and a Welch Xtimate C8 column (4.6 × 250 mm, 3.5 µm). The samples were analyzed under the following gradient of eluent A (8 mM KH2PO4, pH 2.4) and eluent B (100% acetonitril): 5 min 10% B, 20 min linear gradient to 30% B, 3 min linear gradient to 50% B, 5 min 50% B, 2 min linear gradient to 10% B, 5 min 10% B. HMA was detected at 215 nm.