Part:BBa_K5325001
Amy_plaA
An extracellular polylactic acid (PLA) depolymerase taken from the Amycolatopsis sp. able to digest PLA into oligomers and lactic acid monomers.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 637
Illegal PstI site found at 71
Illegal PstI site found at 519 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 637
Illegal PstI site found at 71
Illegal PstI site found at 519 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 637
Illegal PstI site found at 71
Illegal PstI site found at 519 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 637
Illegal PstI site found at 71
Illegal PstI site found at 519 - 1000COMPATIBLE WITH RFC[1000]
Contents
NOTE: As the four parts BBa_K5325000, BBa_K5325001, BBa_K5325002, and BBa_K5325003 were experimented at the same time, experimental methods, results, and figures shown in the four parts' pages will be identical to one another.
Usage and Biology
Poly lactic acid (PLA) depolymerases have been found naturally in several microorganisms and they are able to facilitate digestion of the biopolymer PLA, producing oligomer chains and lactate 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 Amycolatopsis sp. strain K104-1 to allow an engineered bacteria with the part to cleave the PLA polymers to its monomers, which can then be metabolized by the bacteria.
PLA depolymerases can be categorized into 2 types: Type I (proteases) - specific to cleave poly L-lactic acid (PLLA) - and Type II (lipases/cutinases/esterases) - preferentially cleave poly D-lactic acid (PDLA), with BBa_K5325001 matching the description of Type I as the enzyme showed caseinolysis and fibrinolysis activities[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 temperature approaches the hydrolysis temperature of PLA (>50 °C).
Parts Preparation
The four parts BBa_K5325000, BBa_K5325001, BBa_K5325002, and BBa_K5325003 were each assembled into different pRL814 vectors so that only 1 part out of 4 will be in a vector. All parts’ expressions in the pRL814 were controlled by the lac repressor and operon system. Afterwards, the assembled vectors were each transformed into E. coli, and the transformed E. coli are then conjugated with S. oneidensis so that the final result are 4 different S. oneidensis strains and each strain carrying one of the four above-mentioned parts.
SDS-Page and Western Blot Results
<Five S. oneidensis strains that contained empty pRL814 vector, BBa_K5325000, BBa_K5325001, BBa_K5325002, and BBa_K5325003, respectively, were incubated overnight with 100μM IPTG at 30°C before being diluted to OD600nm=1. The resulting dilution was mixed with 0.5 μL of 1 M DTT and was incubated at 95°C for 10 minutes, spun down, and ran through a SDS-PAGE gel. Collected SDS-PAGE gel was then visualized using Western Blot with anti-FLAG antibodies to highlight the proteins of interest in the gel. The Western blot result is shown in Figure 1, which shows the resulting protein band size of Amy_plaA was at 26 kDa. This matched the expected size of the resulting protein when compared to the source article result of PLA depolymerase purified from Amycolatopsis sp. and accounting for the additional C-terminal FLAG tag on the part [2].
Cell Lysate and Cell Supernatant HPLC Analysis
Five S. oneidensis strains with empty pRL814 vector, BBa_K5325000, BBa_K5325001, BBa_K5325002, and BBa_K5325003, respectively, were grown in LB broth cultures overnight before each culture had the supernatant and cells separated into different new LB broth media. All cells were lysed using the freeze-thaw method in which cultures were cooled to -20 °C for 30 minutes before they were heated up to 37 °C; this cycle was repeated for 3 times, theoretically causing cell wall rupture. Supernatant cultures and cell lysate cultures for all strains were incubated with low-molecular weight PLA beads and 100μM IPTG at 37 °C and 0.5 mL from supernatant culture and cell lysate each were taken after 24 hours over a period 5 days. Day 5 culture samples for supernatants and cell lysates of S. oneidensis were analyzed with HPLC for acetate and lactate concentrations. 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 and a high concentration of acetate for all four parts, indicating that the overnight culture had viable S. oneidensis with the capacity to express the parts but the parts were not active in the supernatant culture. On the other hand, in Figure 3, the cell lysates' result for BBa_K5325001 indicated that the sample have the most lactate out of all samples in the treatment group. However, the amount of lactate produced in the negative control pRL814 empty vector was higher than that of all samples in the treatment group. Due to this result, a solid conclusion for the activity of the part BBa_K5325001 cannot be made.
OD600 Analysis
The 5 S. oneidensis strains mentioned previously were grown in M5 minimum broth media supplemented with 200μM Lactate: one set with PLA and the other without PLA as the negative control, and each set having 3 replicates. All replicates were incubated with 100μM IPTG at 30 °C, and OD600 results were taken over the course of 6 days. As the parts were not transported out of the engineered bacteria due to the lack of a Sec system signal sequence, once the broth cultures enter their death phase on day 3, cells will burst open and release their contents, enabling the cytosolic PLA depolymerases to degrade PLA and the remaining cells to metabolize the produced lactate oligomers and monomers to persist longer than cells in cultures with no PLA. The cultures' longevity was predicted to be shown through a higher average OD600 relative to that of cultures with no PLA.
From the result of parametric t-test for paired samples, strain Amy (BBa_K5325001) and ABO (BBa_K5325000) showed statistically 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 persisted longer than cultures without PLA. While the result for BBa_K5325001 culture had the expected pattern of a culture that was able to persist longer due to the presence of active PLA depolymerases, more experimentation with purified part is needed to conclude whether the part is active or not.
Possible Improved Parts
We have designed new parts with an N-terminus PelB signal sequence 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 tested for expression and activity.
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. Nakamura, K., Tomita, T., Abe, N., Kamio, Y. (2001). Purification and Characterization of an Extracellular Poly( l -Lactic Acid) Depolymerase from a Soil Isolate, Amycolatopsis sp. Strain K104-1. Appl Environ Microbiol. 67(1):345-353
//function/degradation
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