|
|
| (14 intermediate revisions by 2 users not shown) |
| Line 5: |
Line 5: |
| | | | |
| | ===Applications of BBa_K4804000=== | | ===Applications of BBa_K4804000=== |
| − | =Introduction=
| |
| − | We have successfully constructed and tested one new composite part (PETase-R, BBa_K4804000). PETase (PET-digesting enzyme) is secreted by a bacterium (Ideonella sakaiensis 201-F6) that has the ability to degrade PET plastic (Harry P., et al., 2017). Spider silk has high strength and toughness, and has a unique three-dimensional spatial network structure, which can be used as a fixed material for some macromolecules.
| |
| − |
| |
| − | =Usage and Biology=
| |
| − | We fused a spider silk protein fragment-R (a repetitive region in pyriform silk gene PySp1) to the C-terminal of PETase (Tuo Yi, 2019). p-NP (p-Nitrophenyl Butyrate) assay results showed that the PETase-R fusion protein could degrade the substrate, and HPLC analysis showed that the PET degradation efficiency was improved. In our project, we used PETase and R to build our biobrick.
| |
| − |
| |
| − | =Characterization=
| |
| − | ==Successful protein expression==
| |
| − | We designed the basic parts (PETase and R) by reading background literature and data, then cloning into pET-21a (+) backbone plasmid by GenScript. The composition part (BBa_K4804000) consist of PETase (BBa_K4804002) and R (BBa_K4804003) (Figure 1). The R sequence was inserted into the C-terminal of PETase using seamless cloning techniques to construct the composition part. In the end, we successfully recombined the PETase-R_pET-21a (+) plasmid, and transformed it into E.coli BL21 (DE3) strain.
| |
| − | The PETase-R expression was detected by SDS-PAGE after induction by IPTG. As shown in the Figure 2, compared to the simple without induction (lane 2), PETase-R induction by IPTG (lanes 3-6) have protein bands near 45 kDa, and the results indicate that the protein has been successfully expressed in BL21(DE3).
| |
| | | | |
| | ===User Reviews=== | | ===User Reviews=== |
Latest revision as of 06:14, 11 October 2023
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_K4804000
User Reviews
UNIQ3f906d6d10e986cc-partinfo-00000000-QINU
UNIQ3f906d6d10e986cc-partinfo-00000001-QINU
