Team:Pasteur Paris/Measurement





Esterase pNP Assay in BAP1


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 BAP1 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 100µL of our bacterial suspension OD(600nm)=0.5 or OD(600nm)=0.1. In the appropriates wells, 10µL 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 (405nm) every 2 minutes. The difference of substrate's concentration or OD(600nm) 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.



1st pNP-Assay

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




BAP1 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.



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