Part:BBa_K5325000

ABO_plaA

An extracellular polylactic acid (PLA) depolymerase that breaks down PLA down to 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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 757
  Illegal AgeI site found at 673
  
• 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 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 ability enables the application of enzymatic degradation for the clean up of environmental PLA, which is not quite as degradable as other types of biodegradable plastics due to its high degradation temperature (>50 °C). With that in mind, our team designed BBa_K5325000 as a candidate of an effective PLA depolymerase part to allow an engineered bacteria with the part to cleave PLA into the corresponding oligomers and monomers, which can then be metabolized by the same bacteria or other bacteria in the environment.

PLA depolymerase, as a group, includes a variety of hydrolases that preferentially target PLA with different compositions, which are poly(D-lactic acid) (PDLA), poly(L-lactic acid) (PLLA), and poly(D,L-lactic acid) (PDLLA); the last of which can have different composition of D-lactic acids and L-lactic acids in its polymer structure. With this in mind, the enzyme group can be categorized into 2 types: proteases - specifically digest PLLA - and lipases/cutinases/esterases - preferentially cleave PDLA over PLLA and is capable of digesting PDLLA^[1]. The original PLA depolymerase that made up BBa_K5325000 fits the description of the latter group as it is an esterase with a preference towards cleaving poly(D,L)-lactate over strictly PLLA or PDLA^[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.

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). Column with the empty vector was used as a negative control for the experiment. All cell lysate samples were treated with DTT to prevent the dimerization of the proteins of interest, and some enzymes that failed to react with DTT remained dimerized and have a higher molecular weight, indicated by the faint band in the RPA column.

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, and the result is shown in Figure 1, with the resulting protein band size of ABO at around 30-35 kDa. This result was 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

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.

The five S. oneidensis strains mentioned above 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. Supernatant cultures and cell lysate cultures 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 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. In addition, the abundance of acetate in the supernatant shown in Figure 2 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. As BBa_K5325000 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, coinciding with the fact that there was no signal sequence on the part that would enable the transport of the part into extracellular space. On the other hand, in Figure 3, the cell lysates' HPLC result for BBa_K5325000 indicated that lactate was produced in the culture, which would have been an indication of PLA depolymerase activity. However, the amount of lactate produced in the negative control pRL814 empty vector was higher than that of BBa_K5325000, thus the lactate production in the cell lysate culture with BBa_K5325000 cannot be concluded to be a result of PLA depolymerase activity.

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 for 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, theoretically allowing the cytosolic PLA depolymerases to degrade PLA and enabling 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, 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 and matching with the prediction stated above. By day 6, there were no significant differences between the average OD₆₀₀ of cultures with PLA and cultures without PLA in all strains, suggesting that all cultures had went through their death phases and there were only very little viable cells left in each culture. This result lent credence to BBa_K5325000 being an active PLA depolymerase. However, due to the lack of extracellular activity of the part, more experiments and concrete results are needed to confirm the part's activity.

Possible Improvements

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. Hajighasemi, M. (2017). Enzymatic Depolymerization of Synthetic Polyesters by Microbial Carboxylesterases. University of Toronto, Toronto, ON.