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. 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 chose 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(600nm)=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 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 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.
| BAP 1 | Average of the measurements | Standard deviation | ||||||||
2nd pNP-Assay
| BAP 1 | |||||||||
| Abs(405 nm) | 0.2072 | 0.1785 | 0.1568 | 0.1541 | 0.0138 | 0.0202 | 0.0093 | 0.0081 | |
| Abs(405 nm) | 0.1885 | 0.1909 | 0.1940 | 0.1393 | 0.0104 | 0.0125 | 0.0148 | 0.0098 | |
3rd pNP-Assay
| BAP 1 | |||||||||
| Abs(405 nm) | 0.1526 | 0.1650 | 0.1648 | 0.1226 | 0.0037 | 0.0071 | 0.0022 | 0.0059 | |
| 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.
Impact of the ethylene glycol and of the terephthalate acid on the strain BAP 1 of E.coli
→ Introduction:
The Plasticure project is based on the capacity of the bacteria of Pfeifer to degrade the polyethylene glycol to produce a medical compound : the erythromycin. To do it, the gene coding for the NB-Esterase enzyme to degrade the polyethylene terephthalate (PET) in ethylene glycol (EG) and in terephtalic acid (TPA) may be used. Unfortunately, the enzyme cited before takes two weeks to degrade a small quantity of polyethylene terephthalate. The products of the degradation are directly used to verify ours metabolic ways. This part will show how was done the processus to deserve the Plasticure project.
→ Devlopment:
The ethylene glycol is toxic when it is too concentrated for the bacteria. So, the aim was to produce a medium where the ethylene glycol is enough concentrated to test ours metabolic ways and do not kill the support BAP1. The terephtalic acid is not soluble in the water and change the pH of its environment. Here, the aim is to produce a medium where BAP1 will be able to use it and survive with a TPA solubilized.
Because we wanted ours owns datas, the needed to test the solubility of the TPA. Because the TPA was known to be soluble in alcohol and DMSO, we wanted to solubilized it and put it in LB to increase it's solubility. The processus failed. So, we tried to change the form of our TPA. We choose the ionic form which is polar and normally hydrophilic and soluble. The bibliography learns us how to use NaOH to produce a ionic form of our TPA : the Na2TPA (disodium terephtalic). When we did it, the reaction was total and the TPA was entirely solubilized. The only one negative point was the basic pH of the solution.
For the EG, it was soluble in water so it's solubility was not a problem.
We decided, after that, to control the toxicity of the EG and the TPA on the support BAP1. It's why we did some culture media with different concentration of those products to test the dose effect. 12 mmol/l was the best concentration we found for TPA in a LB medium, 20 mmol/l for EG but we didn't continue the experiment with EG because the products needed to be in a stoichiometric number to test our future metabolic ways.
We also tried to use the M9 medium, known as a selective medium for E. coli, in order to prove that BAP 1 didn't use EG or TPA as a primary source of carbon. Unfortunately, BAP 1 didn't grow in those media. And, when we tried to control the toxicity of the TPA in LB, we found a better growth with TPA for BAP 1. The pH was not the same in the different media, so we do an other experiment to see if BAP 1 was a basophilic strain of E. coli. This other experiment shows that this hypothesis was wrong for an LB medium. So, we begin to think that BAP 1 could degrade the TPA, and also the fact that our reaction could degrade the TPA in something else that only Na2TPA.
One bibliography was found about the TPAOH (2-hydroxy-terephtalate) which was a fluorescent coumpound. In order to proove that our TPA was solubilised, we do an other reaction with the FeSO4 and H2O2 on our hypothetic Na2TPA. Unfortunately, we found the perfect opposite of what we expected. The solubilised TPA contained more TPAOH before the addition of FeSO4. So, we found that the H2SO4 could precipitate the TPA and the FeSO4 too. It's why it was easy to prove that we have TPA in our solution. But, we needed to prove that TPA becomes also Na2TPA and not only TPAOH which is different of TPA and could act in a different way on BAP 1.
We did a Fluorescence experience to prove that BAP 1 do not use the TPAOH and prove that the reaction between TPA and Na2HPO4 could create Na2TPA. So, our TPA was totally solubilized and present in our medium. A growth experiment done at the same time shows that TPA could be used as a carbon sources by BAP 1. It's only preliminary results, but it could explain why BAP 1 grows better in LB + TPA than LB medium only.
But how we did to product a selective medium for BAP 1? The M9 medium was a fail, it's why we choose to create a M9 medium with vitamins and rare salts before done only a M9 with rare salts. The fact is the M9 with vitamins contains to much carbon source to be a good selective medium. A simple M9 with rare salts (or F1 medium as Pfeifer named it), was better to select the BAP 1, but it's not a total selective medium because our preliminary result shows the growth of some bacteria in this medium.
The EG was also shows as a toxic compound but only for big concentration.
→ Conclution:
The Study of TPA and EG was really interesting because the TPA was first considered as a toxic product and finally as a source of carbon. We can suppose that the toxicity of TPA was the only fact of the pH imposed by the NaOH in excess. The EG appears to be toxic but not to much for our BAP 1 which seems to have a good tolerance to it. The creation of a selective medium was more complicated and it's sad that we can't continue to try to create an other one better than the last we tried. The other regret was about the toxicity of the TPA which is not 12 mmol/l because we can easily imagine that the pH was the cause of the death of the bacteria.
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