Difference between revisions of "Team:BGU ISRAEL/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, thus slowing down their metabolism significantly. Our goal was to use an organism which has adapted into degrading plastic, although slowly, and to examine its mode of action. We choose to work with Rhodococcus ruber C208 which was found to be able to utilize polyethylene (PE) by Prof. Alex Sivan from BGU (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 thus far unreported. We designed an experiment to test the ability of this bacterium to degrade different carbon sources and characterize its growth rate utilizing each carbon source.

PET degradation characterization using electron microscopy:

After our experiments with R. ruber and characterizing how a naturaly evolved plastic degrading bacterium utilizes PET, we turned to examining our bacteria with the same methods.

We incubated 3 PET pellets in a 500ml liquid LB medium with codon-optimzed LC-Cutinase expressing bacteria, to create a large enzyme to substrate ratio.

To ensure that the results are in fact from the action of the LC-Cutinase enzyme we set two controls:

1. LB liquid medium with 3 PET pellets incubated at the same conditions, with no bacteria added.
2. LB liquid medium with 3 PET pellets incubated at the same conditions, with bacteria transformed with the pACYC vector without the Cutinase gene.

The same test was performed using the W.T. LC-Cutinase and F4 variant with PET and PE but the results were inconclusive.
We planned on performing the experiment again, but were unable to a lack of time and resources.

P. putida carbon source utilization experimens:

P. putida has a crucial role in our project’s designs. Our symbiotic approach relies on E. coli utilizing one of PET’s monomer, ethylene glycol, while P. putida, utilizes the other – terephthalate. For P. putida to utilize TPA (terephthalate) it need to be engineered and inserted with a degradation pathway that terminates in protocatechuate (PCA).

P. putida is documented in previously published articles as able to utilize PCA as a sole carbon source (Jiménez et al 2002), but we wanted to verify that, and determine optimal concentrations.

Moreover, as P. putida is known to degrade various toxic molecules we wanted to test it for the degradation of TPA, and possibly eliminate the need for the pathway enegineering.

We used liquid M9 minimal media with either TPA or PCA as carbon sources.

The concentrations of PCA tested were 5 and 10mM and the concentrations of TPA tested were 5,20 and 30mM.

for detailed instruction on media preparation see our Protocols)

References

1. Jiménez, J. I., Miñambres, B., García, J. L., & Díaz, E. (2002). Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440. Environmental microbiology, 4(12), 824-841.

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, by degrading it to its monomer, ethylene glycol and utilizing it 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 hypothesized that if E. coli can express and secrete LC-Cutinase, the enzyme will degrade 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 hypothesis. 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 successfully degrades PET to its respective monomers. We have managed to create a new assay using M9 medium with soft agar plates containing shredded PET.

Bacteria can only grow inside the soft agar if:

1. LC-Cutinase is expressed
2. LC-Cutinase is secreted using the pelB leader sequence
3. 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 occurs as hypothesized.

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.‏

Fuel cell construction and testing:

Designing our fuel cell, we looked for electron mediators that were previously used in MFCs with P. putida KT 2440, in belief that it will improve current generation and power densities (Logan et al 2006). We decided to use Protocatechuic acid (PCA), an intermediate in the terephthalate metabolic pathway we plan to insert into P. putida KT 2440. Furthermore, it was shown that P. putida can utilize PCA, ensuring its ability to permeate through the bacterium's outer membrane (Jimenez et al 2002). We measured its potential via cyclic voltammetry (CV) to assess its compatibility as an electron mediator.

A M9 medium with 1% Protocatechuic acid (20% w/v) as a carbon source was prepared. Potential was measured by cyclic voltammetry. The reference electrode used was an Ag-AgCl electrode, while the working electrode and the counter electrode were 1 mm graphite rods.

After verifying our assumption regarding the electron mediator, we constructed an anodic half-cell:

First, we used graphite 2x2 discs as anodes with P. putida KT 2440 that was induced to form a biofilm onto it a week prior to the experiment. The anode was immersed in 10 ml of LB medium in a 50 ml glass. The media was inoculated with 50 µl from a day old starter of P. putida KT 2440. The sample was incubated in 30OC and stired at 300 RPM. The growth medium had 5 ml of LB replaced with a fresh medium every two days.

After achieving a substantial biofilm growth, an anodic half-cell was assembled. The cell was composed of a 100 ml glass, Ag-AgCl reference electrode, a counter electrode, made of a 0.5 mm graphite rod, a 2x2 graphite disc anode with the biofilm and a 3x2.5 graphite disc cathode. The electrodes were immersed in 45ml of M9 medium with 0.01% Protocatechuic acid and phosphate buffer at pH 7.2. The cells were kept at room temperature on a magnetic plate with a stirrer. The cathode was induced with a potential of 700 mV using a multi-channel potentiostat. For control, a cell without bacteria was also prepared and tested.

Voltage was measured with different resistances ranging from 0-10M Ω using a voltmeter for a period of three days to examine changes in power output and current.

References

1. Logan, B. E., Hamelers, B., Rozendal, R., Schröder, U., Keller, J., Freguia, S., ... & Rabaey, K. (2006). Microbial fuel cells: methodology and technology. Environmental science & technology, 40(17), 5181-5192.
2. Jimenez, J. I., Minambres, B., Garcia, J. L., & Diaz, E. (2002). Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440. Environmental microbiology, 4(12), 824-841.