IGEM:IMPERIAL/2009/Assays Protocols/Trehalose/OtsA

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OtsA : Trehalose –6-Phosphate synthase

TPS Assay : A quantitative assay of the activity of Trehalose –6-Phosphate synthase (units of activity given in μmol of product per minute).

Shopping List

UDPG
G-6-P
Tris-HCl Buffer
MnCl2
heparin salt
HCl
NaOH
anthrone
Shodex Sugar



Alternative Assay

Link to website for assay

Trehalose –6-Phosphate synthase assay : non-radioative method Adapted from Vandercammen et al (Eur. J. Biochem. 182, 613-620., 1989)


Reaction:

Tre6Psynthase:

UDPglucose + Glucose-6-phosphate => Trehalose-6P + UDP

Enzymatic coupling:

Pyruvate kinase: UDP + PEP => Pyruvate + UTP

Lactate Dehydrogenase: Pyruvate + NADH => Lactate + NAD

Preparation of crude extract:

- Break open 15-20 mg dry mass (109 cells) in 0.5 ml extraction buffer (Hepes 20 mM, pH 7.1, DTT 1mM, KCl 100 mM; cocktail inhibitor from Boerhinger)/ 1 g glass beads by vortexing 6 times 30 sec with 30 sec interval in ice.

- Centrifuge tubes at 3000g for 5 min at 4°C

- transfer supernatant into eppendorf tube

- Centrifuge 15min in eppendorf centrifuge at full speed at 4°C


Enzymatic assay:


In 1 ml final volume

           Mixture TPS                                                                                                  0.5 ml 

(Hepes 50 mM, pH 7.1, KCl 100 mM; EDTA 2mM, MgSO4 5 mM)

           UDPglucose 20 mM                                                                                      0.1 ml
           Glc-6-P/ F-6-P 100 mM/30 mM                                                                   0.1 ml
           Water                                                                                                            0.2 ml
           Sample                                                                                               0.1 ml

Incubate at 42°C

After 10, 20, 30 min

Take 0.25 ml in 0.75 ml of ice-cold UDP mixture kept in ice-cold water bath

Measure OD340 (E0)

Add Lactate dehydrogenase 100 U/ml 1 µl

Read OD340 (E1)

Add Pyruvate kinase 100U/ml 1 µl

Read OD340 (E2)


Calcul of activity:

Tre6PS activity (in nmol UDP produced/ min / mg protein) is given by:

D (Es2-Es1) – (D Ebk2-Ebk1)/ 6.22 x 1000/100 x 1000/4 x 1/time x 1/protein (mg/ml)


With E is value of OD for the sample and Ebk value OD for the blank

The blank is carried out with the extract that has been heat inactivated for 5 min at 80°C before addition to the mixture.

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