Difference between revisions of "Team:BGU ISRAEL/Experiments"
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an ability that was previously unknown. We designed an experiment to test the ability of this bacterium to degrade different carbon sources and characterize its growth rate utilizing each one. | an ability that was previously unknown. We designed an experiment to test the ability of this bacterium to degrade different carbon sources and characterize its growth rate utilizing each one. | ||
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and that it manages to degrade the PET to its monomers. We have managed to create a new assay using M9 medium with soft agar plates containing shredded PET. | and that it manages to degrade the PET to its monomers. We have managed to create a new assay using M9 medium with soft agar plates containing shredded PET. | ||
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Using various assays and substrates to examine the variants gave a deep comprehensive understanding regarding the variants and their degradation activity. | Using various assays and substrates to examine the variants gave a deep comprehensive understanding regarding the variants and their degradation activity. | ||
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Revision as of 09:44, 16 October 2016
Experiments
Rhodococcus ruber plastic degradation assays:
Since plastic is a new synthetic polymer introduced to the environment by mankind only recently, not many organisms have adapted into using plastic as a sole carbon source. Furthermore, organisms that are able to utilize plastic still consume it in an inefficient manner, making their metabolism rate slow. Our goal was to use an organism which has adapted into degrading plastic at some level and to examine the methods it uses. We choose to work with Rhodococcus ruber C208 which was found to be able to utilize polyethylene (PE) (Orr et al 2004).
Because our project focuses on the biodegradation of PET, and not PE, we first wanted to test Rhodococcus ruber’s ability to utilize PET as a sole carbon source, an ability that was previously unknown. We designed an experiment to test the ability of this bacterium to degrade different carbon sources and characterize its growth rate utilizing each one.
Terephthalate acid presence assay:
LC-Cutinase degrades PET into two products, ethylene glycol and terephthalic acid (TPA).
We wanted to check for the presence of TPA and to measure the kinetics of the LC-Cutinase variants using the levels of TPA.
To do that we conducted an experiment using previously used essay (Barreto et al 1994), but reversed the usage of the essay to find the concentration of TPA .
We prepared different concentrations of TPA and irradiated them with UV. By measuring emissions we were able to make a calibration curve for TPA concentrations.
We then took samples from a M9 medium containing Escherichia coli that secretes LC-Cutinase to check for the presence of TPA after the degradation of PET and measure the kinetics of the degradation reaction.
Barreto, J. C., Smith, G. S., Strobel, N. H., McQuillin, P. A., & Miller, T. A. (1994). Terephthalic acid: a dosimeter for the detection of hydroxyl radicals in vitro. Life sciences, 56(4), PL89-PL96.
Examining the utilization of PET by E. coli expressing LC-Cutinase:
After testing the LC-Cutinase (wild type, codon optimized and variant genes) ability to degrade pNP-Butyrate, we also wanted to examine the possibility that by expressing the enzyme, our E. coli strain (BL21) has gained the ability to utilize PET as a sole carbon source.
During our review of existing literature regarding plastic degradation and the degradation of its monomers, we learned that most E. coli strains have the ability to consume ethylene glycol, one of the PET degradation products (Boronat, A. et. al, 1983). We theorized that if E. coli can express and secrete LC-Cutinase, the enzyme will degrade the PET to TPA (terephthalic acid) and EG (ethylene glycol), and the bacteria will be able to consume EG and grow without any other carbon source.
We needed to develop an assay that will test this theory. Our goal was to build a relatively simple experiment that could confirm the fact that LC-Cutinase is expressed and secreted by the E. coli, and that it manages to degrade the PET to its monomers. We have managed to create a new assay using M9 medium with soft agar plates containing shredded PET.
The bacteria can only grow inside the soft agar if:
- LC-Cutinase is expressed
- LC-Cutinase is secreted using the pelB leader sequence
- LC-Cutinase can degrade the PET to EG and TPA
If one of the conditions in this process is not met, there should not be any growth of E. coli on PET. This assay gave us a simple way to confirm that the expression, secretion and degradation of LC-Cutinase are happening as planned.
Boronat, A., Caballero, E., & Aguilar, J. (1983). Experimental evolution of a metabolic pathway for ethylene glycol utilization by Escherichia coli. Journal of bacteriology, 153(1), 134-139.
pNP-Butyrate degradation assay:
The pNP-B degradation assay is a fast and simple method to estimate LCC’s (LC-Cutinase) variants’ degradation activity. Under the tested conditions, LCC has a high pNP-B substrate specificity. During the degradation of the pNP-B by LCC, pNP is released, the quantity of which can be simply and accurately determined via measuring absorption at 405nm. This method, allows a quick scan of many variants’ degradation activity in different conditions, and the determination of said variants’ kinetic constants. The experiments were conducted in room temperature, since the enzyme is designated for use in similar conditions and because the enzyme is known to function well in room temperature.
Due to the similarity between the bond degraded by LCC in PET and pNP-B, LCC’s effectiveness in degrading PET can be assessed by its efficiency in pNP-B degradation. With that in mind, since PET has a more complex chemical structure (it is a polymer), this method alone is obviously not enough to absolutely determine the enzyme’s efficiency with PET as a substrate. For this reason, pNP-B degradation assay was used as a preliminary scan of the different variants and as method to determine their kinetic constants, alongside PET degradation assays. Using various assays and substrates to examine the variants gave a deep comprehensive understanding regarding the variants and their degradation activity.
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