Difference between revisions of "Part:BBa K5136023"

 
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K5136023 short</partinfo>
 
<partinfo>BBa_K5136023 short</partinfo>
  
−
1
+
===Biology===
 +
OleT<sub>JE</sub> from Jeotgalicoccus sp. ATCC 8456 is the first identified P450 fatty acid decarboxylase, which efficiently catalyzes a single-step decarboxylation of FFAs to form α-olefins by consuming H<sub>2</sub>O<sub>2</sub> (as sole oxygen and electron donor) stoichiometrically (1).
 +
<br/>It can cause alkaline fracture of conjugated side chains of lignin and other colored substances such as azo dyes through nucleophilic reaction, increasing the hydrophilicity of the reaction products, which can be easily removed in the subsequent washing process to achieve the purpose of bleaching (2).
 +
===Usage and design===
 +
OleT<sub>JE</sub> can cause alkaline fracture of conjugated side chains of lignin and other colored substances such as azo dyes through nucleophilic reaction, increasing the hydrophilicity of the reaction products, which can be easily removed in the subsequent washing process to achieve the purpose of bleaching (2).
 +
 
 +
 
 +
===Construction===
 +
We use pET-28b(+) to construct this circuit. Then the ligation mixture was transformed into <i>E. coli</i> DH5α & <i>E. coli</i> BL21(DE3), and the positive transformants were confirmed by kanamycin, colony PCR, and sequencing.
 +
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/haoxihuan/bba-k4907128-hrpr.png" width="400px"></html></center>
 +
<center><b>Figure 1 Gene circuit of His tag-<i>OLET<sub>JE</sub></i>.</b></center>
 +
 
 +
====Routine Characterization====
 +
When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1521 bp
 +
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/haoxihuan/bba-k4907128-hrpr.png" width="400px"></html></center>
 +
<center><b>Figure 2 DNA gel electrophoresis of the colony PCR products of BBa_K5136013_pET-28a(+).</b></center>
 +
 
 +
The plasmid verified by sequencing was successfully transformed into <i>E. coli BL21(DE3). After being cultivated and induced by 0.5 mM IPTG at 20 °C, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. SDS-PAGE and Coomassie blue staining were used to verify the expression of the target protein (about 48.4 kDa).
 +
 
 +
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/haoxihuan/bba-k4907128-hrpr.png" width="400px"></html></center>
 +
<center><b>Figure 3 SDS-PAGE analysis of CYP199A4 T253L-His tag protein.</b></center>
 +
 
 +
===Deinking Experiments===
 +
 
 +
 
 +
===Reference===
 +
1. Jiang, Y., Li, Z., Zheng, S. et al. Establishing an enzyme cascade for one-pot production of α-olefins from low-cost triglycerides and oils without exogenous H<sub>2</sub>O<sub>2</sub> addition. <i>Biotechnol Biofuels</i> <b>13</b>, 52 (2020).
 +
<br/>2. Shen Kui-zhong, Application of Hydrogen Peroxide in the Pulp and Paper Industry. (2005).
 +
 
  
−
<!-- Add more about the biology of this part here
 
−
===Usage and Biology===
 
  
 
<!-- -->
 
<!-- -->

Revision as of 15:29, 30 September 2024

OleTJE

Biology

OleTJE from Jeotgalicoccus sp. ATCC 8456 is the first identified P450 fatty acid decarboxylase, which efficiently catalyzes a single-step decarboxylation of FFAs to form α-olefins by consuming H2O2 (as sole oxygen and electron donor) stoichiometrically (1).
It can cause alkaline fracture of conjugated side chains of lignin and other colored substances such as azo dyes through nucleophilic reaction, increasing the hydrophilicity of the reaction products, which can be easily removed in the subsequent washing process to achieve the purpose of bleaching (2).

Usage and design

OleTJE can cause alkaline fracture of conjugated side chains of lignin and other colored substances such as azo dyes through nucleophilic reaction, increasing the hydrophilicity of the reaction products, which can be easily removed in the subsequent washing process to achieve the purpose of bleaching (2).


Construction

We use pET-28b(+) to construct this circuit. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3), and the positive transformants were confirmed by kanamycin, colony PCR, and sequencing.

Figure 1 Gene circuit of His tag-OLETJE.

Routine Characterization

When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1521 bp

Figure 2 DNA gel electrophoresis of the colony PCR products of BBa_K5136013_pET-28a(+).

The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by 0.5 mM IPTG at 20 °C, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. SDS-PAGE and Coomassie blue staining were used to verify the expression of the target protein (about 48.4 kDa).

Figure 3 SDS-PAGE analysis of CYP199A4 T253L-His tag protein.

Deinking Experiments

Reference

1. Jiang, Y., Li, Z., Zheng, S. et al. Establishing an enzyme cascade for one-pot production of α-olefins from low-cost triglycerides and oils without exogenous H2O2 addition. <i>Biotechnol Biofuels 13, 52 (2020).
2. Shen Kui-zhong, Application of Hydrogen Peroxide in the Pulp and Paper Industry. (2005).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 715
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 715
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 715
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 715
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 715
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 667