Difference between revisions of "Part:BBa K3185005"
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A 3µL of protein solution dropped on PET film, then left for 20min.  Then the film was washed in TBST for 5min x3, then placed with Anti-His-tag-HRP conjugated for 1h.  ECL substrate was added, then chemiluminescence was imaged by LAS-3000.  The exposure time is 6min.
 
A 3µL of protein solution dropped on PET film, then left for 20min.  Then the film was washed in TBST for 5min x3, then placed with Anti-His-tag-HRP conjugated for 1h.  ECL substrate was added, then chemiluminescence was imaged by LAS-3000.  The exposure time is 6min.
 
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[[File:Fiber CBB.png|300px|thumb|right|Fig.3 SDS-PAGE gel for quantification of amounts of proteins bind to PET fiber
 
[[File:Fiber CBB.png|300px|thumb|right|Fig.3 SDS-PAGE gel for quantification of amounts of proteins bind to PET fiber
 
20cm of PET fibers were soaked in protein solutions, then washed in TBST for 5min three times.  Washed fibers were soaked in 50µL of 2x SDS sample buffer.  Bounded proteins were eluted with boiling. SDS-PAGE for 40min in 200V. CBB stained.
 
20cm of PET fibers were soaked in protein solutions, then washed in TBST for 5min three times.  Washed fibers were soaked in 50µL of 2x SDS sample buffer.  Bounded proteins were eluted with boiling. SDS-PAGE for 40min in 200V. CBB stained.
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As shown in Fig.2, the negative control protein, SpyCatcher (SPYC), did not stain PET film at all. In contrast, all the plastic-binding proteins tested here strongly stained the PET film. Although BaCBM2 and CenA might be darker than the other two proteins, as stains spread, we could not quantify their signals. This blot spreading might be due to the plastic-binding proteins’ fast binding rate. The proteins in excess liquid could have bound to the neighbor area of the film in the first wash step.
 
As shown in Fig.2, the negative control protein, SpyCatcher (SPYC), did not stain PET film at all. In contrast, all the plastic-binding proteins tested here strongly stained the PET film. Although BaCBM2 and CenA might be darker than the other two proteins, as stains spread, we could not quantify their signals. This blot spreading might be due to the plastic-binding proteins’ fast binding rate. The proteins in excess liquid could have bound to the neighbor area of the film in the first wash step.
  
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Although this experiment suggested our plastic-binding proteins can quickly bind to PET’s smooth surface, we could not compare binding affinity quantitatively.<br><br>
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Although this experiment suggested our plastic-binding proteins can quickly bind to PET’s smooth surface, we could not compare binding affinity quantitatively.
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<br><br>
 
<h3><font size="4.5">PET fiber assay</font> </h3>
 
<h3><font size="4.5">PET fiber assay</font> </h3>
 
We showed two fluorescent plastic-binding proteins bind to PET cloth very tightly. Next, we demonstrated proteins’ binding in a more realistic target: PET fiber. In cloth, fibers are close to each other, so they might create a hydrophobic environment between them. In the fiber form, they are surrounded by water, so plastic-binding proteins might behave in a different way. <br><br>
 
We showed two fluorescent plastic-binding proteins bind to PET cloth very tightly. Next, we demonstrated proteins’ binding in a more realistic target: PET fiber. In cloth, fibers are close to each other, so they might create a hydrophobic environment between them. In the fiber form, they are surrounded by water, so plastic-binding proteins might behave in a different way. <br><br>

Revision as of 15:47, 21 October 2019


SPYCatcher -> CenA W68Y

Usage and Biology

CBM-CenA is a Carbohydrate-binding Module from cellulase of Cellulomonas fimi. Originally, it has binding affinity to cellulose, but its binding ability to PET is enhanced because of point mutation [1]. In the paper, they compare many kinds of mutants and it is found that a mutant whose #68 amino acid W is changed by Y (CenA W68Y) has the most strong binding affinity to PET.

We used it for PET binding domain. We put SpyCatcher on the N-terminus of CenA W68Y because we used SpyCatcher/SpyTag system to bind it to other parts(SpyCatcher:BBa_K1159200, SpyTag:BBa_K1159201). In addition, this has three tag and cleavage sites. First is inserted 6×His-tag in N-terminus of SpyC to purify. Second is inserted MYC-tag between SpyCather and CenA to detect it by using the antibody. Third is inserted TEV protease site because, in the paper, it is used for protein purification. However, we didn’t use it in our experiment.

We put it between BamHI site and Ndel site on pET11-a. The expression plasmids were introduced into BL21(DE3) and expressed by T7 promoter/ T7 RNAP system. Ni-NTA agarose was used for the purification.

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 AgeI site found at 751
    Illegal AgeI site found at 841
  • 1000
    COMPATIBLE WITH RFC[1000]

Purification

Fig.1 SDS-PAGE of imidazole elutes, CBB stained


Expression

  • Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37oC shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.
  • Protein was expressed in 0.1mM IPTG for 2hours.

Purification

1. E. coli which expressed this part were lysed with sonification.
2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).
3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.

This purification method works. As shown in Fig.1, the protein successfully purified.


Result

Fig.2 Plastic-binding protein binding to PET film
A 3µL of protein solution dropped on PET film, then left for 20min. Then the film was washed in TBST for 5min x3, then placed with Anti-His-tag-HRP conjugated for 1h. ECL substrate was added, then chemiluminescence was imaged by LAS-3000. The exposure time is 6min.
Fig.3 SDS-PAGE gel for quantification of amounts of proteins bind to PET fiber 20cm of PET fibers were soaked in protein solutions, then washed in TBST for 5min three times. Washed fibers were soaked in 50µL of 2x SDS sample buffer. Bounded proteins were eluted with boiling. SDS-PAGE for 40min in 200V. CBB stained.

PET film assay

We tried to compare our proteins with each other by the film dot blotting.

As shown in Fig.2, the negative control protein, SpyCatcher (SPYC), did not stain PET film at all. In contrast, all the plastic-binding proteins tested here strongly stained the PET film. Although BaCBM2 and CenA might be darker than the other two proteins, as stains spread, we could not quantify their signals. This blot spreading might be due to the plastic-binding proteins’ fast binding rate. The proteins in excess liquid could have bound to the neighbor area of the film in the first wash step.

Although this experiment suggested our plastic-binding proteins can quickly bind to PET’s smooth surface, we could not compare binding affinity quantitatively.

PET fiber assay

We showed two fluorescent plastic-binding proteins bind to PET cloth very tightly. Next, we demonstrated proteins’ binding in a more realistic target: PET fiber. In cloth, fibers are close to each other, so they might create a hydrophobic environment between them. In the fiber form, they are surrounded by water, so plastic-binding proteins might behave in a different way.

As shown in Fig.3, CenA is a polyethylene terephthalate (PET)-binding protein. According to the references, CenA is a polyethylene terephthalate (PET)-binding protein. Therefore, this result is consistent with the reported observation.

References

1 Zhang, Y., Chen, S., Xu, M., Cavoco-Paulo, A.P., Wu, J., and Chen, J. (2010).
Characterization of thermobifida fusca cutinase-carbohydrate-binding module fusion proteins and their potential application in bioscouring∇.
Appl. Environ. Microbiol. 76, 6870–6876.

Zhang, Y., Wang, L., Chen, J., and Wu, J. (2013).
Enhanced activity toward PET by site-directed mutagenesis of Thermobifida fusca cutinase-CBM fusion protein.
Carbohydr. Polym. 97, 124–129.