Difference between revisions of "Part:BBa K3763023"

 
 
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This part can be induced to express our interested protein Propionicin T1 with native peptide and 6xHis-tag in cytosol,which can be purified using Ni-IMAC.After purification, we can used the extracts to test the inhibition ability of PctA against Propionibacterium acnes.Further more,we designed that the native leader peptide can guide the secretion of PctA in engineered bacteria.
 
This part can be induced to express our interested protein Propionicin T1 with native peptide and 6xHis-tag in cytosol,which can be purified using Ni-IMAC.After purification, we can used the extracts to test the inhibition ability of PctA against Propionibacterium acnes.Further more,we designed that the native leader peptide can guide the secretion of PctA in engineered bacteria.
  
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==Results==
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===Usage and Biology===
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After constructing BBa_K3763023 on plasmid pET26b+, we transformed the expression vector into BL21(DE3) and designed a orthogonal test to find the best condition for induced PctA expression. The expression of PctA was induced with IPTG in a set of different conditions and checked with SDS-PAGE, and the amount of protein was estimated by the gray value of the band. The result is as follows.
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                <p style="color:Gray; padding:0px 30px 10px;">Figure 1 SDS-PAGE of BL21 with expression vector induced in different conditions, lysate supernatant (A) and precipitation (B). 0: uninduced BL21(DE3); 1-9: induced group, lane 1-9. Arrow indicates the band of precursor PctA. </p>
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<br>Results showed that 11kDa precursor PctA is successfully induced in the bacteria, but no band of 7kDa mature PctA is observed in the culture media, which meant that PctA was not successfully secreted. As shown in Fig.3 (A) and (B), after induction, the pellet of bacteria lysate contained most of the precursor PctA, while the lysate supernatant had a much smaller portion. Through the orthogonal test, we found that there were a maximum of total PctA expression at 30℃, 1mM IPTG, 3h. To get more PctA in the supernatant, this condition was further optimized into 30℃, 0.5mM IPTG, 4h.
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Latest revision as of 02:26, 22 October 2021


the expression operator in pET26b+ and CDS of PctA

This part can be induced to express our interested protein Propionicin T1 with native peptide and 6xHis-tag in cytosol,which can be purified using Ni-IMAC.After purification, we can used the extracts to test the inhibition ability of PctA against Propionibacterium acnes.Further more,we designed that the native leader peptide can guide the secretion of PctA in engineered bacteria.

Results

After constructing BBa_K3763023 on plasmid pET26b+, we transformed the expression vector into BL21(DE3) and designed a orthogonal test to find the best condition for induced PctA expression. The expression of PctA was induced with IPTG in a set of different conditions and checked with SDS-PAGE, and the amount of protein was estimated by the gray value of the band. The result is as follows.

Figure 1 SDS-PAGE of BL21 with expression vector induced in different conditions, lysate supernatant (A) and precipitation (B). 0: uninduced BL21(DE3); 1-9: induced group, lane 1-9. Arrow indicates the band of precursor PctA.


Results showed that 11kDa precursor PctA is successfully induced in the bacteria, but no band of 7kDa mature PctA is observed in the culture media, which meant that PctA was not successfully secreted. As shown in Fig.3 (A) and (B), after induction, the pellet of bacteria lysate contained most of the precursor PctA, while the lysate supernatant had a much smaller portion. Through the orthogonal test, we found that there were a maximum of total PctA expression at 30℃, 1mM IPTG, 3h. To get more PctA in the supernatant, this condition was further optimized into 30℃, 0.5mM IPTG, 4h.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 356
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 340