Difference between revisions of "Part:BBa K3759019"
 
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LCC is a leaf-branch compost cutinase[1] and a kinetically robust protein[2]. A research published on Nature came up with a mutant enzyme, mLCC[1] that hydrolyzes 90% of PET in plastic bottles in just 10 hours. This is more efficient than any previous PET hydrolase, and more importantly, the resulting monomers- ethylene glycol and terephthalic acid have the same properties as the monomers found in petrochemical materials.
 
LCC is a leaf-branch compost cutinase[1] and a kinetically robust protein[2]. A research published on Nature came up with a mutant enzyme, mLCC[1] that hydrolyzes 90% of PET in plastic bottles in just 10 hours. This is more efficient than any previous PET hydrolase, and more importantly, the resulting monomers- ethylene glycol and terephthalic acid have the same properties as the monomers found in petrochemical materials.
  
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The linker is GSGSGS.
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The linker is GGGGSGGGGS.
  
 
BslA is a structurally defined bacterial hydrophobin that was found in the biofilm of Bacillus subtilis.  
 
BslA is a structurally defined bacterial hydrophobin that was found in the biofilm of Bacillus subtilis.  
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1. a 6× His tag is added to enable us carrying out Ni-NTA protein purification.
 
1. a 6× His tag is added to enable us carrying out Ni-NTA protein purification.
  
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===Protein Expression===
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===Protein Purification===
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Because the pET28a vector has His-tag, therefore we used affinity chromatography to purify the supernatant obtained after bacteria clastogenesis using nickel. The result of our proteogels after elution with 200 mm imidazole and 300 mm imidazole is shown below. We successfully purified mLCC and mLCC-linker-BslA. At the same time, we can also see that the two proteins, mLCC-mHGFI, mLCC-mHFBI, are expressed, but the protein expression level is very low.
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 +
[[File:mlcc10.png|center|400px]]<br>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
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https://2021.igem.org/wiki/images/9/9e/T--BJEA_China--protein_expression.jpg<br>
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'''Figure 1.'''(a). M: marker; <br>
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'''Figure 1.''' The expression of mLCC-linker-BslA (Left 1st 2nd)
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Lane 1, 2: mLCC-linker-BslA 43kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;<br>
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</p >
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Lane 3, 4: mLCC 28kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;<br>
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Lane 5, 6: mLCC-linker-mHFBI 36kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;<br>
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Lane 7, 8: mLCC-linker-mHGFI 36kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
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</p>
  
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Pre-expression:
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===PET Degradation Reaction===
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We perform PET degradation reaction to detect enzyme activity. We set several protein concentration to detect enzyme activity, under the condition of pH8, 70℃, and 18h of reaction time. Then we measured the absorption value at uv240nm by nanodrop, which is the absorption position of the product TPA, and we were surprised to find that the relative enzyme activity of mLCC-linker-BslA was increased about 3 times compared to mLCC!
  
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The BL21 bacteria that contains aimed protein were cultured in 5mL LB liquid medium with kanamycin in 37℃ overnight. After taking samples, we transfer them into 1L LB medium with kanamycin.
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<p style="text-align: center;">
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(a)[[File:mlcc11.png|400px]]<br>
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Cultured in bottles:
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(b)[[File:mlcc12.png|400px]]<br>
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'''Figure 2.''' (a). The adsorption value of mLCC, mLCC-Linker-BslA of UV240 under the condition of pH8, 70 celsius degree, reaction time of 18h.<br>
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After culturing in 37℃ in bottles, we used 0.5mM IPTG induced in 16℃ for 24 hours. Then, we used 200mM imidazole to eluting and get left 1st aimed protein, and we used 300mM imidazole to eluting the left 2nd aimed protein.
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(b) The relativity activity of mLCC, mLCC-Linker-BslA.<br></p>
  
 
===References===
 
===References===
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[3]: “BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm.” Proceedings of the National Academy of Sciences of the United States of America vol. 110,33 (2013): 13600-5. doi:10.1073/pnas.1306390110  
 
[3]: “BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm.” Proceedings of the National Academy of Sciences of the United States of America vol. 110,33 (2013): 13600-5. doi:10.1073/pnas.1306390110  
  
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<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 12:29, 20 October 2021


mLCC-linker-BsLA

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 261
    Illegal EcoRI site found at 1079
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 261
    Illegal EcoRI site found at 1079
    Illegal NheI site found at 193
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 261
    Illegal EcoRI site found at 1079
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 261
    Illegal EcoRI site found at 1079
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 261
    Illegal EcoRI site found at 1079
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage

It has been well known that the surface of PET film is hydrophobic, and the surface of mLCC is hydrophilic. By constructing the mLCC-linker-BsLA fusion protein, the PET degradation efficiency will be enhanced enormously, due to the unique properties of amphiphilicity and self-assembly of hydrophobin BslA. Also, as BslA was extracted from bacteria and was a bacterial hydrophobin, it shows a better fusion with mLCC, which help the increment of the PET degradation efficiency of mLCC-linker-BslA.

Biology

LCC is a leaf-branch compost cutinase[1] and a kinetically robust protein[2]. A research published on Nature came up with a mutant enzyme, mLCC[1] that hydrolyzes 90% of PET in plastic bottles in just 10 hours. This is more efficient than any previous PET hydrolase, and more importantly, the resulting monomers- ethylene glycol and terephthalic acid have the same properties as the monomers found in petrochemical materials.

The linker is GGGGSGGGGS.

BslA is a structurally defined bacterial hydrophobin that was found in the biofilm of Bacillus subtilis. It helps the assembling of TasA (an exopolysaccharide and an amyloid fiber-forming protein), the component of the biofilm matrix. BslA is composed of an Ig-type fold with the addition of an unusual, extremely hydrophobic “cap” region. The central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. [3]

Design Consideration

The construct was cloned into a pET28a plasmid and transformed into BL21 (DE3) E. coli.

The construction includes:

1. a 6× His tag is added to enable us carrying out Ni-NTA protein purification.

Protein Purification

Because the pET28a vector has His-tag, therefore we used affinity chromatography to purify the supernatant obtained after bacteria clastogenesis using nickel. The result of our proteogels after elution with 200 mm imidazole and 300 mm imidazole is shown below. We successfully purified mLCC and mLCC-linker-BslA. At the same time, we can also see that the two proteins, mLCC-mHGFI, mLCC-mHFBI, are expressed, but the protein expression level is very low.

Mlcc10.png

Figure 1.(a). M: marker;
Lane 1, 2: mLCC-linker-BslA 43kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 3, 4: mLCC 28kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 5, 6: mLCC-linker-mHFBI 36kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 7, 8: mLCC-linker-mHGFI 36kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;

PET Degradation Reaction

We perform PET degradation reaction to detect enzyme activity. We set several protein concentration to detect enzyme activity, under the condition of pH8, 70℃, and 18h of reaction time. Then we measured the absorption value at uv240nm by nanodrop, which is the absorption position of the product TPA, and we were surprised to find that the relative enzyme activity of mLCC-linker-BslA was increased about 3 times compared to mLCC!

(a)Mlcc11.png
(b)Mlcc12.png
Figure 2. (a). The adsorption value of mLCC, mLCC-Linker-BslA of UV240 under the condition of pH8, 70 celsius degree, reaction time of 18h.
(b) The relativity activity of mLCC, mLCC-Linker-BslA.

References

[1] Tournier, V. , Topham, C. M. , Gilles, A. , David, B. , & Marty, A. . (2020). An engineered pet depolymerase to break down and recycle plastic bottles. Nature, 580(7802), 216-219.

[2] Sulaiman S , You D J , Kanaya E , et al. Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-Cutinase[J]. Biochemistry, 2014, 53(11):1858-1869.

[3]: “BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm.” Proceedings of the National Academy of Sciences of the United States of America vol. 110,33 (2013): 13600-5. doi:10.1073/pnas.1306390110