Team:Pasteur Paris/Measurement







Esterase pNP Assay in BAP 1


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. Est13 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 chose a different substrate of Est13: the 4-Nitrophenyl butyrate, also called para-Nitrophenylbutyrate. The 4-Nitrophenyl butyrate has a similar chemical structure compared to PET, but is way smaller.


→ Protocol:

In a P96 plate, we had in each well 100 µl of our bacterial suspension OD(600nm)=0.5 or OD(600nm)=0.1. In the appropriate wells, 10 µl of susbtrate at 10 mM, 50 mM, or 0 mM was added. The plate was incubated at 34°C in the spectrophotometer for 30 minutes. We use a TECAN spectrophotometer for this experiment: we took the respective suspension's absorbance (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 BAP 1 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 substrate, to verify whether our results come really from the esterase enzymatic activity;

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



1st pNP-Assay

For this first experiment, we started the measurement of the absorbance 1 hour after the addition of the substrate.




BAP 1 Average of the measurements Standard deviation
C1
C2
C3
C-
C1
C2
C3
C-
0mM of substrate
Abs(405nm)
0.1345
0.1227
0.1249
0.1267
0.0027
0.0009
0.0861
0.0018
10 mM of Substrate
Abs(405nm)
1.1567
0.9178
0.3750
0.2688
0.0407
0.1712
0.0000
0.0018



2nd pNP-Assay

BAP 1
Average of the measurements
Standard deviation
C1
C2
C3
C-
C1
C2
C3
C-
0mM of substrate
 Abs(405 nm) 0.2072 0.1785 0.1568 0.1541 0.0138 0.0202 0.0093 0.0081
10mM of Substrate
Abs(405 nm) 0.1885 0.1909 0.1940 0.1393 0.0104 0.0125 0.0148 0.0098



3rd pNP-Assay



BAP 1
Average of the measurements
Standard deviation
C1
C2
C3
C-
C1
C2
C3
C-
0 mM of substrate
 Abs(405 nm) 0.1526 0.1650 0.1648 0.1226 0.0037 0.0071 0.0022 0.0059
10 mM of substrate
 		Abs(405 nm) 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 BAP 1 strain to degrade the substrate 4-Nitrophenyl Butyrate. The negative control shows that BAP 1 itself doesn't degrade 4-Nitrophenyl butyrate naturally. The observed accumulation of 4-Nitrophenol Butyrate most likely depends on the Esterase plasmid.



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