Difference between revisions of "Part:BBa K3185006"

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[[File:191001 SPYt-SPYC 2.png|300px|thumb|left|Fig. 10 Isopeptide bond formation between Plastic binding proteins and Encapsulin.
 
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3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature.  Then 6µL of 2x SDS sample buffer was added.  10µL of each sample was loaded.  SDS-PAGE for 30min in 200V.  The gel was CBB stained.
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The dilution collection of each protein was dropped on PET cloth, then left for 20min.  The protein fluorescent was imaged by LAS-3000.  The exposure time is 10sec.]]
 
The dilution collection of each protein was dropped on PET cloth, then left for 20min.  The protein fluorescent was imaged by LAS-3000.  The exposure time is 10sec.]]
 
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3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature.  Then 6µL of 2x SDS sample buffer was added.  10µL of each sample was loaded.  SDS-PAGE for 30min in 200V.  The gel was CBB stained.
 
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==References==  
 
==References==  

Revision as of 09:58, 19 October 2019


SPYCatcher -> sfGFP -> LCI KR-2

Usage and Biology

LCI is a protein from Bacillus subtili. The paper shows that it can bind to polypropylene(PP)[1]. The paper shows the improved variant, LCI-KR2(Y29R and G35R; variant KR-2)[2]. Its affinity is 5.4±0.5 times stronger than natural LCI.

We used LCI-KR2 for binding protein to PP. We inserted superfolder GFP (sfGFP) whose folding interval is shortened by improving natural GFP on the N-terminus of LCI (BBa_I746916). By doing so we wanted to do the binding assay with fluorescence. Moreover, we put SpyCatcher(BBa_K1159200)[ on N-terminus of sfGFP because we used SpyTag/SpyCatcher system to bind it to other parts.

This part has four tags. First is 6×His-tag inserted on the N-terminus of SpyC for protein purification. Second is MYC-tag inserted between sfGFP and Spy-Catcher to detect it by using the antibody. The third is a TEV protease site and we put it into two regions because it was used for protein purification in the paper[3].

We inserted it into the site between the BamHI site on the pGEX11-a and Ndel site. We used BL21 (DE3) for gene expression. We used Ni-NTA Agarose for purification. After that, we confirmed the molecular weight of LCI by using SDS-PAGE. The result is shown below.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1174
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 460

Purification


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.

SDS-PAGE

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Result

Fig.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.



File:Cloth dot blot washed.png
Fig. Cloth dot blot by fluorescent plastic-binding protein before washing.
The dilution collection of each protein was dropped on PET cloth, then left for 20min. The protein fluorescent was imaged by LAS-3000. The exposure time is 10sec.



Fig. 10 Isopeptide bond formation between Plastic binding proteins and Encapsulin.

3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature. Then 6µL of 2x SDS sample buffer was added. 10µL of each sample was loaded. SDS-PAGE for 30min in 200V. The gel was CBB stained.



Fig.3a Cloth dot blot by fluorescent plastic-binding protein before washing. The dilution collection of each protein was dropped on PET cloth, then left for 20min. The protein fluorescent was imaged by LAS-3000. The exposure time is 10sec.


References

1 Rübsam, K., Stomps, B., Böker, A., Jakob, F., and Schwaneberg, U. (2017).
Anchor peptides: A green and versatile method for polypropylene functionalization.
Polymer (Guildf). 116, 124–132.

2 Rübsam, K., Davari, M.D., Jakob, F., and Schwaneberg, U. (2018).
KnowVolution of the polymer-binding peptide LCI for improved polypropylene binding.
Polymers (Basel). 10, 1–12.

3 Rübsam, K., Weber, L., Jakob, F., and Schwaneberg, U. (2018).
Directed evolution of polypropylene and polystyrene binding peptides.
Biotechnol. Bioeng. 115, 321–330.