Difference between revisions of "Part:BBa K3759020"

 
 
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<partinfo>BBa_K3759020 short</partinfo>
 
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===Usage and Biology===
 
  
 
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<partinfo>BBa_K3759020 parameters</partinfo>
 
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===Usage===
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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 to enhance the PET degradation efficiency of mLCC-linker-BslA.
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===Biology===
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BslA is a structurally defined bacterial hydrophobin that was found in the biofilm of Bacillus subtilis.
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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. [1]
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The linker is GGGGSGGGGS.
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===Design Consideration===
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The construct was cloned into a pET28a plasmid and transformed into BL21 (DE3) E. coli.
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The construction includes:
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 +
1. a 6× His tag is added to enable us carrying out Ni-NTA protein purification.
 +
 +
===Protein Expression===
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<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.''' The expression of mLCC-linker-BslA (Left 1st 2nd)
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</p >
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Pre-expression:
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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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Cultured in bottles:
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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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===References===
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[1]: “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
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===Usage and Biology===

Latest revision as of 03:22, 20 October 2021


linker-BsLA

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 294
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 294
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 294
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 294
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 294
  • 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 to enhance the PET degradation efficiency of mLCC-linker-BslA.

Biology

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. [1]

The linker is GGGGSGGGGS.

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 Expression

T--BJEA_China--protein_expression.jpg
Figure 1. The expression of mLCC-linker-BslA (Left 1st 2nd)

Pre-expression:

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.

Cultured in bottles:

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.

References

[1]: “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