Part:BBa_K5117048

PcotYZ-BsRBS-AtCelO-L2-CotY-B0014

This part serves as transcriptional unit composed of:

• promoter PcotYZ of Bacillus subtilis (BBa_K5117021)
• ribosome binding site of Bacillus subtilis (BBa_K5117000)
• celO gene of Acetivibrio thermocellus without signal peptide encoding an exoglucanase (EC 3.2.1.176),
addition of a long flexible linker (L2) downstream of the coding sequence encoding the amino acids (GGGGS)₄ (BBa_K5117047)
• cotY gene of Bacillus subtilis (BBa_K5117022)
• bidirectional terminator B0014 (BBa_B0014)

Biosafety level: S1

Target organism: Bacillus subtilis

Main purpose of use: Immobilization of AtCelO on the spore crust of B. subtilis (spore surface display)

Application: Degradation of cellulose

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1184
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2170
  
• 1000
  COMPATIBLE WITH RFC[1000]

Spore preparation

Spores were prepared by culturing cells in LB medium with chloramphenicol until they reached the exponential growth phase (OD₆₀₀ of 0.4–0.6). After washing and resuspension in DSM, the culture was incubated at 37 °C for 24 hours to induce sporulation. The cells were then lysed using lysozyme and washed with dH₂O and SDS to remove vegetative cell residues. For qualitative plate assays, an OD₆₀₀ of 0.2 was used. Further details are available on the Experimentspage.

Detemination of exoglucanase activity with PASC assays and PASA assay

We attempted to determine exoglucanase activity using various methods, including assays with avicel and congo red staining, as well as a cellulose overlay assay. Despite multiple iterations, these approaches were unsuccessful. Ultimately, we established the PASA and PASC assays based on work of Percival Zhang et al. (2006), which allowed for clear visualization of exoglucanase activity through the appearance of clearance zones, as shown in Fig. 6, without the need for additional staining. Details of the assay development can be found on the Engineeringpage.

Fig. 6: PASC and PASA assays for determination of exoglucanase activity. The arrow indicates the zone of clearance, which signifies exoglucanase activity. The sample used was 20 µL of commercially available cellobiohydrolase I, and the plates were incubated at 50 °C for 24 hours.

To examine the functionality of AtCelO-displaying spores (AtCelO-L2), a qualitative assay was performed using PASC-Agar and PASA-Agar plates, following the protocol described on the Experimentspage. Initially, the optical density (OD) of the spore solution was adjusted to 0.2, and 20 µL was pipetted onto the plates. The plates were then incubated at 50 °C for 24 hours, as shown in Figure 7 (example of a PASC assay). However, no activity was detected. Further testing with spore solutions at OD₆₀₀ ranging from 0.5 to 4.0, as well as incubation at room temperature and 65 °C, also showed no activity (data not shown).

Fig. 7: Qualitative PASC activity assay of AtCelO displaying spores (AtCeO-L2) on PASC-agar plate to examine cellobiohydrolase activity. OD₆₀₀ of the spore solution was adjusted to 0.2 and 20 µL of the suspension was applied to PASC-agar plates.

References

Percival Zhang, Y. H., Cui, J., Lynd, L. R., & Kuang, L. R. (2006). A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: Evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules, 7(2), 644–648.