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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 757
Illegal AgeI site found at 673 - 1000COMPATIBLE WITH RFC[1000]
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 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 Alcanivorax borkumensis (A. borkumensis) to allow an engineered bacteria with the part to cleave the PLA polymers to its monomers, which can then be metabolized by the bacteria.
SDS-Page and Western Blot Results
S. oneidensis culture was diluted to OD600nm=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 using Western Blot with anti-FLAG antibodies to highlight Amy_plaA on the gel. Gel result is shown in Figure 1, with BBa_K5325000 result being shown with 3 additional parts: BBa_K5325001, BBa_K5325002, BBa_K5325003, and an empty pRL814 vector acting as a negative control. The resulting protein band size of Amy_plaA was at 26 kDa, which is expected 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.
Activity
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
//function/degradation
| biology | Alcanivorax borkumensis |