Difference between revisions of "Part:BBa K5325000"

Revision as of 04:09, 2 October 2024

ABO_plaA

An extracellular polylactic acid (PLA) depolymerase that breaks down PLA down to oligomers and lactic acid monomers.

Sequence and Features

Usage and Biology

PLA depolymerases can be found naturally in a few microorganisms, and they are able to facilitate the natural digestion of the biopolymer PLA, producing lactate oligomer chains and monomers. This makes the protein a promising option for the clean up of PLA waste in the environment, which is not quite as biodegradable as the plastic was advertised to be and is still capable of persisting and polluting natural habitats. With that in mind, our team designed this part as a candidate of an effective PLA depolymerase part that was taken from Alcanivorax borkumensis (A. borkumensis) to allow an engineered bacteria with the part to cleave PLA polymers to the corresponding oligomers and monomers, which can then be metabolized by bacteria.

PLA depolymerases can be categorized into 2 types: proteases - specific to cleave poly L-lactic acid (PLLA) - and lipases/cutinases/esterases - preferentially cleave poly D-lactic acid (PDLA) over PLLA, with BBa_K5325000 fitting with the description of the latter as the PLA depolymerase of origin is an esterase with a preference towards cleaving poly(D,L)-lactate (PDLL) over strictly PLLA or PDLA^[1,2]. While the mechanism of how PLA depolymerase binds to and hydrolyze PLA is still unknown, PLA depolymerase hydrolysis activity is known to increase as the temperature approaches the hydrolysis temperature of PLA (>50 °C).

SDS-Page and Western Blot Results

S. oneidensis culture was diluted to OD_600nm=1, then the culture was incubated at 95°C and spun down before the resulting cell lysate was run through the SDS-PAGE gel. Collected SDS-PAGE gel was then visualized through Western Blot with anti-FLAG antibodies to highlight the proteins of interest, which included BBa_K5325000 (ABO),BBa_K5325001 (Amy),BBa_K5325002 (RPA), and BBa_K5325003 (Pa). Gel result is shown in Figure 1, with the resulting protein band size of ABO being at around 30-35 kDa, which is expected when compared to the theorized size of ABO2449 - the original PLA depolymerase that is the primary part of BBa_K5325000 - accounting for the additional C-terminal FLAG tag on the part^[2].

Cell Lysate and Cell Supernatant HPLC Analysis

S. oneidensis in LB broth culture with BBa_K5325000 was grown overnight before the supernatant and cells were separated into different LB broth media. Cells were lysed using the freeze-thaw method in which the cell cultures were subjected to cooling to -20 °C for 30 minutes before being heated to 37 °C for 3 times consecutively. Supernatant cultures and cell lysate cultures were incubated with low-molecular weight PLA beads at 37 °C and 0.5 mL from supernatant culture and cell lysate each were taken after 24 hours for 5 days. Day 5 culture samples for the supernatant and cell lysate of S. oneidensis containing BBa_K5325000, BBa_K5325001, BBa_K5325002, BBa_K5325003, and the empty vector pRL814 was analyzed with HPLC for acetic acid contents and lactic acid contents. Lactate and acetic acid concentrations in Day 5 sample for the supernatant and cell lysate cultures of S. oneidensis containing the four parts and empty vector are shown in Figure 2 and Figure 3, respectively.

In Figure 2, the supernatants' HPLC result does not show any lactate content for all four parts, including BBa_K5325000. As the part was expected to cleave PLA to produce the monomer lactic acid, the result shown here indicate that the part was likely non-functional in the extracellular space. The abundance of acetate in the supernatant is likely a result of S. oneidensis metabolism in LB broth culture, indicating that the cell was growing and thus should have been able to express the part. On the other hand, in Figure 3, the cell lysates' HPLC result for ABO (BBa_K5325000) indicated that lactate was produced in the culture. However, the amount of lactate produced in the negative control pRL814 empty vector was higher than that of ABO, thus the lactate production in the cell lysate culture with BBa_K5325000 cannot be concluded to be a result of PLA depolymerase activity

OD₆₀₀Analysis

Another experiment was performed on all 4 strains, namely by growing them in M5 minimum media supplemented with 200uM Lactate, one set with PLA and the other without PLA for control. Since our enzymes cannot be transported out of the cell due to the lack of PelB sequence, OD₆₀₀ was taken over the course of 6 days. Since cells enter their death phase at around day 3, they will burst open and release their contents, theoretically the PLA depolymerase can then degrade PLA and the remaining cells can survive better than in an environment with no PLA.

Strain Amy (BBa_K5325001) and ABO (BBa_K5325000) showed significant differences between cultures with PLA and cultures without PLA while the OD₆₀₀ values of all cultures showed a significant decrease from the 48 hours time point to 72 hours time point, suggesting that Amy and ABO cultures with PLA presence have better maintenance than cultures without PLA.

This matches the hypothesis - as the engineered S. oneidensis cultures enter their death phase, cells undergo more lysis and release the PLA depolymerase trapped inside the cell's cytosol. These enzymes can then start digesting the PLA beads down to oligomers and lactate monomers inside cultures with the PLA beads. The lactate monomers can be consumed and used by the remaining cells within the cultures and allow the cells in the culture to be maintained for a longer period (in theory), resulting in a relatively higher OD₆₀₀ value relative to cultures w/o PLA. Following this idea, cultures with PLA should maintain a higher OD₆₀₀ until PLA is exhausted, or when the PLA digestion rates become insufficient for the maintenance of the cultures with PLA beads. However, by day 6, there are no significant differences - most cultures with PLA have an OD₆₀₀ roughly similar to that of cultures without PLA (Amy, ABO, and RPA), suggesting that both cultures have undergone their death phase and there are only very little viable cells left in each culture. As such, the differences in BBa_K5325000 culture growth cannot be concluded to be a result of PLA depolymerase activity.

Possible Improved Parts

We have designed new parts with an N-terminus PelB tag for periplasmic secretion and a C-terminus His tag for more affordable extraction columns. However, as of September 16th 2024, these new parts have not been successfully transformed into E. coli and thus cannot be subject to more experiments.

References

1. Kawai, F., Nakadai, K., et al. (2011). Different enantioselectivity of two types of poly(lactic acid) depolymerases toward poly(l-lactic acid) and poly(d-lactic acid). Polym. Degrad. Stab. 96(7):1342-1348.
2. Hajighasemi, M., Nocek, B.P., et al. (2016). Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases. Biomacromolecules. 17(6):2027-2039