Difference between revisions of "Part:BBa K1921003"
 
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===Usage===
 
===Usage===
−
The PET-ase is an enzyme, which can hydrolyze PET. And this mutation protein is changed on the basis of the PET–ase. This protein is changed from R to T at 64 position. This mutation protein can hydrolyze PET in low temperature, which is a big problem in the world. It also can increase in the bacteria, which is easy to get.
+
The PETase is an enzyme, which can hydrolyze PET. And this mutation protein is changed on the basis of the PETase. This protein is changed from S to F at 181 position. On the one hand, this mutation protein can hydrolyze PET in low temperature, which is a big problem in the world. On the other hand, it can increase the activity in the high concentration to the native protein. This mutation can continue high activity more times than native. It also can increase in the bacteria, which is easy to get. <br>
  
 
===Biology===
 
===Biology===
−
Compared with the sequence of PETase in NCBI, the homology of PETase and Enzyme TfCut2 was 52%. The paper says that scientists Ren Wei found that change TfCut2 enzyme amino acid 63 T into other amino acids, the degradation products of the reaction will inhibit the reaction, and the vicinity amino acid sequence at the site of TfCut2 enzyme has a high homology with PETase. Based on the literatures, we decided to mutate the amino acid R at this position to T for lower inhibition from product.
+
Compared with the sequence of PETase in NCBI, the homology of PETase and Enzyme Est119 was 50%. The paper says that scientists Kengo Kitadokoro found that the phenylalanine at position 248 is the key site for the flexible binding of this enzyme to carbon chains of different lengths, and the vicinity amino acid sequence at the site of Enzyme Est119 has a high homology with PETase. Based on the literatures, we decided to mutate the amino acid S at this position to F To expand the hydrophobic response pocket (polar amino acids to non-polar amino acids).<br>
  
 
===Protein Expression===
 
===Protein Expression===
−
The bacteria were cultured in 5mL LB liquid medium with ampicillin in 37℃ 5h. After taking samples, we used 1mM IPTG 5μl induced in 16℃ for 4-5h. Then we make the expression .
+
The bacteria were cultured in 5mL LB liquid medium with ampicillin in 37℃ 5h. After taking samples, we used 1mM IPTG 5μl induced in 16℃ for 4-5h. Then we make the expression .
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/d/d9/Tjuresults3.jpg<br>
 +
</p>
 +
'''Figure 1.'''The result of pre-expression of pET-21b-MutateD/J/M and pET-21b-PETase.“+” is induced with IPTG,“-” is not induced with IPTG. <br>
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/6/63/Tjuresults4.jpg<br>
 +
</p>
 +
'''Figure 2.'''The result of the purification of PETase and its 3 mutants. The 4 kinds of protein are purified trough nickel columns. <br>
 +
 
 +
 
 +
===HPLC Result===
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/2/22/ProofTJU1.jpg<br>
 +
'''Figure 3.'''The Comparison of the enzyme activity between PETase and three kinds of mutated PETase. The reaction condition is 100μL solution,pH 9.0(bicine-NaOH), 40 degree, 18h, the substrate is a round with a diameter of 2mm. The results are detected by Hplc. The y-axis stands for the area of the peak of MHET, the main product of the PETase’s degrading of PET. The x-axis stands for the concentration of the protein. <br>
 +
</p>
 +
 
 +
===Improvement===
 +
<html>
 +
<div class="figure">
 +
<img src="https://2019.igem.org/wiki/images/c/cb/T--TU_Kaiserslautern--Goldkriteriumhoffentlich.png"/>
 +
<p class="caption"><span class="phat">Cytosolic expression of PETase (BBa_K1921003) in Chlamydomonas</span>
 +
                (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequence for HA-tagged PETase (Mutate M) (b) UVM4 transformants containing construct L2AH were grown in TAP medium for four days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW-molecular weight. Transformant J6 served as positive control. Untransformed UVM4 parent strain served as negative control. 
 +
            </p>
 +
</div>
 +
 
 +
We improved expression and activitiy of the PETase, <a href="https://parts.igem.org/Part:BBa_K1921003">BBa_K1921003</a>, by finely modifying its expression in Chlamydomonas and significantly improving its PET degradation efficiency by building <a href="https://parts.igem.org/Part:BBa_K3002014">BBa_K3002014</a>.
 +
</html>

Latest revision as of 03:50, 22 October 2019


PETase MutateM


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage

The PETase is an enzyme, which can hydrolyze PET. And this mutation protein is changed on the basis of the PETase. This protein is changed from S to F at 181 position. On the one hand, this mutation protein can hydrolyze PET in low temperature, which is a big problem in the world. On the other hand, it can increase the activity in the high concentration to the native protein. This mutation can continue high activity more times than native. It also can increase in the bacteria, which is easy to get.

Biology

Compared with the sequence of PETase in NCBI, the homology of PETase and Enzyme Est119 was 50%. The paper says that scientists Kengo Kitadokoro found that the phenylalanine at position 248 is the key site for the flexible binding of this enzyme to carbon chains of different lengths, and the vicinity amino acid sequence at the site of Enzyme Est119 has a high homology with PETase. Based on the literatures, we decided to mutate the amino acid S at this position to F To expand the hydrophobic response pocket (polar amino acids to non-polar amino acids).

Protein Expression

The bacteria were cultured in 5mL LB liquid medium with ampicillin in 37℃ 5h. After taking samples, we used 1mM IPTG 5μl induced in 16℃ for 4-5h. Then we make the expression .

Tjuresults3.jpg

Figure 1.The result of pre-expression of pET-21b-MutateD/J/M and pET-21b-PETase.“+” is induced with IPTG,“-” is not induced with IPTG.

Tjuresults4.jpg

Figure 2.The result of the purification of PETase and its 3 mutants. The 4 kinds of protein are purified trough nickel columns.


HPLC Result

ProofTJU1.jpg
Figure 3.The Comparison of the enzyme activity between PETase and three kinds of mutated PETase. The reaction condition is 100μL solution,pH 9.0(bicine-NaOH), 40 degree, 18h, the substrate is a round with a diameter of 2mm. The results are detected by Hplc. The y-axis stands for the area of the peak of MHET, the main product of the PETase’s degrading of PET. The x-axis stands for the concentration of the protein.

Improvement

Cytosolic expression of PETase (BBa_K1921003) in Chlamydomonas (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequence for HA-tagged PETase (Mutate M) (b) UVM4 transformants containing construct L2AH were grown in TAP medium for four days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW-molecular weight. Transformant J6 served as positive control. Untransformed UVM4 parent strain served as negative control.

We improved expression and activitiy of the PETase, BBa_K1921003, by finely modifying its expression in Chlamydomonas and significantly improving its PET degradation efficiency by building BBa_K3002014.