Part:BBa_K5325003

Pa_TB13_plaA

An extracellular polylactic acid (PLA) depolymerase able to digest PLA into oligomers and lactic acid monomers.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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 are hydrolases that catalyze the digestion of the biopolymer PLA, resulting in lactate oligomers 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. Based on this concept, BBa_K5325003 was designed as a PLA depolymerase part originating from Paenibacillus amylolyticus (P. amylolyticus) strain TB-13. This part, once transferred into a Gram-negative bacteria, would theoretically allow the engineered bacteria to cleave PLA polymers in its environment to the corresponding lactate oligomers and monomers, which can then be metabolized by the same bacteria or other organisms.

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)^[1]. BBa_K5325003, based on the experiment results of the original enzyme described in a previous study, matching the description of Type I as the enzyme showed caseinolysis and fibrinolysis activities^[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

Figure 1: Western Blot of cell lysate from S. oneidensis cell with each carrying the empty vector pRL814, BBa_K5325000 (ABO),BBa_K5325001 (Amy),BBa_K5325002 (RPA), and BBa_K5325003 (Pa). All cell lysate samples were treated with DTT to prevent the dimerization of the proteins of interest. Column with the empty vector pRL814 was used as a negative control for the experiment.

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 OD_600nm=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 through Western Blot with anti-FLAG antibodies. The Western blot result is shown in Figure 1, which showed the resulting protein band size of Pa_TB13_plaA at 17-19 kDa. This closely matched the expected size of the part, based on the molecular weight result of PlaA that was expressed in E. coli, which was 22 kDa, from a previous study^[2].

Cell Lysate and Cell Supernatant HPLC Analysis

Figure 2: Concentration of lactate and acetate in supernatant culture of S. oneidensis expressing BBa_K5325003 (Pa), empty vector pRL814, BBa_K5325000 (ABO), BBa_K5325002 (RPA), and BBa_K5325001 (Amy). The molecules' concentrations were measured at mM.

Figure 3: Concentration of lactate and acetate in culture with S. oneidensis lysate containing BBa_K5325003(Pa), empty vector pRL814, BBa_K5325000(ABO), BBa_K5325002(RPA), and BBa_K5325001(Amy). The molecules' concentrations were measured at mM.

Five S. oneidensis strains that contained 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 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 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 for 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 samples for the supernatant and cell lysate cultures of S. oneidensis 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_K5325003. As the part was expected to cleave PLA to produce the monomer lactic acid, the result shown here indicate that the part of interest was likely non-functional in the extracellular space. Abundance of acetate in all supernatant samples were likely a result of S. oneidensis metabolism in LB broth cultures, indicating that cells were growing and thus all the cells in the respective samples should have been able to express the parts. On the other hand, in Figure 3, the cell lysates' HPLC result for RPA 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 ABO, which should have not been the case as the cell lysate negative control had no way of generating lactate in the LB media. Due to this result, a solid conclusion for the activity of the part BBa_K5325003 in the cell lysate samples 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 at 30 °C with 100μM IPTG, 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.

Figure 4: Average OD₆₀₀ values of BBa_K5325003(Pa), empty vector pRL814, BBa_K5325000(ABO), BBa_K5325002(RPA), and BBa_K5325001(Amy) cultures with PLA (stitched lines) and cultures without PLA (solid lines) over 6 days. OD₆₀₀ of all cultures was measured on day 1, 2, 3, and 6 of the experiment period, shown in the figure as 4 data points per graph.

From the result of parametric t-test for paired samples, the strain with Pa (BBa_K5325003) did not show a statistically significant difference between the OD₆₀₀ values of cultures with PLA and cultures without PLA at all time points. From this result, it is likely that the cytosolic part BBa_K5325003 is not active in the cell.

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. Akutsu-Shigeno, Y., Teeraphatpornchai, T., et al. (2003). Cloning and Sequencing of a Poly( dl -Lactic Acid) Depolymerase Gene from Paenibacillus amylolyticus Strain TB-13 and Its Functional Expression in Escherichia coli. Appl Environ Microbiol. 69(5):2498-2504.