Difference between revisions of "Part:BBa K3715062"

 
 
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<partinfo>BBa_K3715062 parameters</partinfo>
 
<partinfo>BBa_K3715062 parameters</partinfo>
 
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===Usage and Biology===
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This composite part is made up with five basic parts, the RBS, two cutting sites BamHI and XhoI (linker d,e) , the His tag,and our target protein PETase_MT4. It encodes a protein which is PETase_MT4 fused with His-Sumo tag. The fusion protein is about 31.49kD. In order to gain the highly purified target protein, we add His tag and Sumo tag in N-terminal of PETase_MT4 and combine the two parts with the cutting site of protease. The fusion protein can be cut off at the cutting site by protease. It is convenient for us to purify our target protein.
 +
 +
===Origin(organism)===
 +
Ideonella sakaiensis
 +
===Molecular cloning===
 +
First, we used the vector pET28b_SUMO 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;">
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[[File:MT28b1 10.png|500px]]<br>
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'''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-SUMO 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:28bProtein1-12.png|500px]]<br>
 +
'''Figure 2.'''  The result of SDS-PAGE.<br>
 +
</p>
 +
Well, we can know that the expression level of PETase_MT4 on the vector(pET28b_SUMO) is  low，from the result of SDS-PAGE (Figure2) . Therefore, we change the vector and tag to obtain high-expression protein. (If you want to know more about this, please see our project introduction. )<br>
 +
===Conclusion===
 +
The vector we used (pET28b_SUMO) did not meet our requirements well, so we replaced the vector and tag.

Latest revision as of 05:31, 1 October 2021


T7 promoter+RBS+ His+Linker f+SUMO+Linker d+PETase_MT4+Linker e+T7 terminator

This part consists of T7 promoter, RBS, protein coding sequence(His+Linker f+Sumo+Linker d+PETase_MT4+ Linker e) and T7 terminator,and the biological module can be build into E.coli for protein expression.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 296
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 296
    Illegal NheI site found at 73
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 296
    Illegal BglII site found at 185
    Illegal BamHI site found at 1285
    Illegal XhoI site found at 384
    Illegal XhoI site found at 1271
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 296
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 296
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with five basic parts, the RBS, two cutting sites BamHI and XhoI (linker d,e) , the His tag,and our target protein PETase_MT4. It encodes a protein which is PETase_MT4 fused with His-Sumo tag. The fusion protein is about 31.49kD. In order to gain the highly purified target protein, we add His tag and Sumo tag in N-terminal of PETase_MT4 and combine the two parts with the cutting site of protease. The fusion protein can be cut off at the cutting site by protease. It is convenient for us to purify our target protein.

Origin(organism)

Ideonella sakaiensis

Molecular cloning

First, we used the vector pET28b_SUMO to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

MT28b1 10.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-SUMO 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.

28bProtein1-12.png
Figure 2. The result of SDS-PAGE.

Well, we can know that the expression level of PETase_MT4 on the vector(pET28b_SUMO) is low，from the result of SDS-PAGE (Figure2) . Therefore, we change the vector and tag to obtain high-expression protein. (If you want to know more about this, please see our project introduction. )

Conclusion

The vector we used (pET28b_SUMO) did not meet our requirements well, so we replaced the vector and tag.