Difference between revisions of "Part:BBa K4724021"
Line 7: Line 7:
 
A recombinant plasmid containing this composite element was constructed using pET-22b(+) as the carrier. IsPETase and hydrophobic domain CBM3 with Linker were fused to obtain recombinant plasmids by Gibson assembly. Transform the plasmid into the competent state of E. coli BL21 (DE3). Colony PCR such as Fig.1 was performed with T7 and the posterior primers of the amplification domain as the anterior and posterior primers, respectively.
 
A recombinant plasmid containing this composite element was constructed using pET-22b(+) as the carrier. IsPETase and hydrophobic domain CBM3 with Linker were fused to obtain recombinant plasmids by Gibson assembly. Transform the plasmid into the competent state of E. coli BL21 (DE3). Colony PCR such as Fig.1 was performed with T7 and the posterior primers of the amplification domain as the anterior and posterior primers, respectively.
 
<img src="https://static.igem.wiki/teams/4724/wiki/1-fig-1.png" >
 
<img src="https://static.igem.wiki/teams/4724/wiki/1-fig-1.png" >
 +
222222
  
  

Revision as of 10:35, 10 October 2023


IsPETase-Linker-CBM3-6xHisTag

The hydrophobic nature of the PET surface will hinder IsPETase from binding PET, resulting in IsPETase not working well. We add the hydrophobic domain CBM3 to IsPETase, and under the force of water molecule movement, IsPETase connected to the hydrophobic domain will more easily bind to the PET hydrophobic surface.

Construction process

A recombinant plasmid containing this composite element was constructed using pET-22b(+) as the carrier. IsPETase and hydrophobic domain CBM3 with Linker were fused to obtain recombinant plasmids by Gibson assembly. Transform the plasmid into the competent state of E. coli BL21 (DE3). Colony PCR such as Fig.1 was performed with T7 and the posterior primers of the amplification domain as the anterior and posterior primers, respectively. <img src="1-fig-1.png" > 222222


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 56
    Illegal PstI site found at 933
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 56
    Illegal PstI site found at 933
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 790
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 56
    Illegal PstI site found at 933
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 56
    Illegal PstI site found at 933
    Illegal NgoMIV site found at 891
    Illegal AgeI site found at 546
  • 1000
    COMPATIBLE WITH RFC[1000]