Difference between revisions of "Part:BBa K3715004"
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<partinfo>BBa_K3715004 parameters</partinfo>
 
<partinfo>BBa_K3715004 parameters</partinfo>
 
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 +
===Origin(organism)===
 +
Ideonella sakaiensis
 +
===Molecular cloning===
 +
First, we used the vector pET21a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
 +
<p style="text-align: center;">
 +
[[File:SuperDNA.png|300px]]<br>
 +
'''Figure 1.'''  The verification results by enzyme digestion.<br>
 +
</p>
 +
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.
 +
 +
===Expression and purification===
 +
'''Pre-expression:'''<br>
 +
The bacteria were cultured in 5mL LB liquid medium with ampicillin(50μg/mL) in 37℃ overnight.<br>
 +
'''Massive expressing:'''<br>
 +
After taking samples, we transfered them into 900ml LB medium and added antibiotic to 50 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 5-6 hours). Induce the culture to express protein by adding 0.5 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.<br>
 +
 +
'''Affinity Chromatography:'''<br>
 +
We used the Ni Agarose to purify the target protein. The Ni Agarose can combine specifically with the Ni-His tag fused with target protein. <br>
 +
* First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.<br>
 +
* Second, add the protein solution to the column, let it flow naturally and bind to the column. <br>
 +
* Third, add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.<br>
 +
* Forth,add Ni-Washing buffer several times. Check as above. Collect the eluted proteins for further operation.<br>
 +
 +
<p style="text-align: center;">
 +
[[File:SuperProtein.png|300px]]<br>
 +
'''Figure 2.'''  The result of SDS-PAGE.<br>
 +
</p>
 +
'''Gel filtration chromatography:'''<br>
 +
The collected protein samples are concentrated in a 30 KD concentrating tube at a speed of 3400 rpm and concentrated for a certain time until the sample volume is 500 μl. At the same time, the superdex 75 column is equilibrated with a buffer to balance 1.2 column volumes. The sample is then loaded and 1.5 cylinders are eluted isocratically with buffer. Determine the state of protein aggregation based on the peak position and collect protein samples based on the results of running the gel.<br>
 +
 +
<p style="text-align: center;">
 +
[[File:Supe4gel.png|300px]]<br>
 +
 +
'''Figure 3.'''  The result of gel filtration used the superdex75 column with the AKTA system.<br>
 +
</p>
 +
===Enzyme activity determination===
 +
We use HPLC equipment to measure the peak area of the product of PET(MHET) of the reaction, in order to express the enzyme activity of PETase. For more information on the product of PET(MHET), please see our project introduction.<br>
 +
 +
<p style="text-align: center;">
 +
[[File:Supe4e.png|300px]]<br>
 +
 +
'''Figure 4.'''  Enzyme activity determination, compared with wild type.
 +
</p>
 +
===Conclusion===
 +
In conclusion,the enzyme activity and thermostability of Supe4 has greatly improved ，compared with WT(wild type).

Revision as of 02:11, 1 October 2021


Super4

PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbeIdeonella sakaiensis. Well, Super4 is a complicated mutation of PETase, which contains 11 mutation sites：S214H-I168R-W159H-S188Q-R280A-A180I-G165A-Q119Y-L117F-T140D-A80V.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 783
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 783
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 783
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 783
    Illegal NgoMIV site found at 58
    Illegal NgoMIV site found at 112
    Illegal NgoMIV site found at 139
  • 1000
    COMPATIBLE WITH RFC[1000]


Origin(organism)

Ideonella sakaiensis

Molecular cloning

First, we used the vector pET21a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

SuperDNA.png
Figure 1. The verification results by enzyme digestion.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.

Expression and purification

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(50μg/mL) in 37℃ overnight.
Massive expressing:
After taking samples, we transfered them into 900ml LB medium and added antibiotic to 50 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 5-6 hours). Induce the culture to express protein by adding 0.5 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.

Affinity Chromatography:
We used the Ni Agarose to purify the target protein. The Ni Agarose can combine specifically with the Ni-His tag fused with target protein.

  • First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.
  • Second, add the protein solution to the column, let it flow naturally and bind to the column.
  • Third, add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  • Forth,add Ni-Washing buffer several times. Check as above. Collect the eluted proteins for further operation.

SuperProtein.png
Figure 2. The result of SDS-PAGE.

Gel filtration chromatography:
The collected protein samples are concentrated in a 30 KD concentrating tube at a speed of 3400 rpm and concentrated for a certain time until the sample volume is 500 μl. At the same time, the superdex 75 column is equilibrated with a buffer to balance 1.2 column volumes. The sample is then loaded and 1.5 cylinders are eluted isocratically with buffer. Determine the state of protein aggregation based on the peak position and collect protein samples based on the results of running the gel.

300px
Figure 3. The result of gel filtration used the superdex75 column with the AKTA system.

Enzyme activity determination

We use HPLC equipment to measure the peak area of the product of PET(MHET) of the reaction, in order to express the enzyme activity of PETase. For more information on the product of PET(MHET), please see our project introduction.

300px
Figure 4. Enzyme activity determination, compared with wild type.

Conclusion

In conclusion,the enzyme activity and thermostability of Supe4 has greatly improved ，compared with WT(wild type).