Difference between revisions of "Team:Pasteur Paris/Measurement"

In order to make a strain that combines PET degradation pathway and Erythromycin synthesis pathway we investigated the activity of the Esterase (Est13) in the BAP 1 E. coli strain, used for Ery production in Pfeifer’s laboratory. Esterase is the first enzyme in PET degradation pathway, unfortunately the reaction between PET and Esterase is very slow, it takes approximately 2 weeks to accumulate detectable degradation products. To bypass this technical difficulty we sect a different substrate of Esterase: the 4-Nitrophenyl butyrate, also called para-Nitrophenylbutyrate. The 4-Nitrophenyl Butyrate have a similar chemical structure with the PET, but is a way smaller.

→ Protocol:

In a P96 plate, we had in each well 100 µl of our bacterial suspension OD(600 nm)=0.5 or OD(600nm)=0.1. In the appropriates wells, 10 µl of susbtrate 10mM, 50mM, or 0mM was added. The plate was put incubating at 34°C in the spectrophotometer for 30 minutes. We use a spectrophotometer TECAN for this experiment: we took the respective suspension's absorptions (405 nm) every 2 minutes. The difference of substrate's concentration or OD(600 nm) help us to know which conditions are optimal for this enzymatic activity.

→ Our Controls:

We did 3 controls for this experiment:

• With only the medium and the substrate, to see if there are a reaction between this 2 products;
• With the unmodified BAP1 bacteria in presence of the substrate, to see if the bacteria degrade the 4-Nitrophenyl butyrate without our construct;
• With the modified bacteria or unmodified bacteria without substrat, too see that our results come really from the esterase enzymatic activity;

We realized this experiment 3 times for more precision, and with 3 differents clones of our construction.

For this 1st experiment, we started the measurement of the Absorption 1 hour after the addition of the substrate.

BAP 1			Average of the measurements				Standard deviation
		C1	C2	C3	C-	C1	C2	C3	C-
OmM of substrate	Abs(405nm)	0.1345	0.1227	0.1249	0.1267	0.0027	0.0009	0,0861	0.0018
10mM of Substrate	Abs(405nm)	1,1567	0,9178	0,3750	0,2688	0,04066	0,1712	0	0,0018

2nd pNP-Assay

BAP1		Average of the measurements				Standard deviation
		C1	C2	C3	C-	C1	C2	C3	C-
OmM of substrate	Abs(405nm)	0.2072	0.1785	0.1568	0.1541	0.0138	0.0202	0.0093	0.0081
10mM of Substrate	Abs(405nm)	0.1885	0.1909	0.1940	0.1393	0.0104	0.0125	0.0148	0.0098

3rd pNP-Assay

BAP1		Average of the measurements				Standard deviation
		C1	C2	C3	C-	C1	C2	C3	C-
OmM of substrate	Abs(405nm)	0.1526	0.1650	0.1648	0.1226	0.0037	0.0071	0.0022	0.0059
10mM of Substrate	Abs(405nm)	0.1727	0.1797	0.1437	0.1337	0.0294	0.0054	0.0034	0.0095

→ Conclusion

Presence of the plasmid containing the gene of Esterase allows BAP1 strain to degrade the substrate 4-Nitrophenyl Butyrate. The negative control shows that BAP1 itself doesn't degrade 4-Nitrophenyl Butyrate naturally. The observed accumulation of 4-Nitrophenol Butyrate most likely depends on the Esterase plasmid.