Difference between revisions of "Part:BBa K4195035"
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After transforming the plasmid into ''E. coli''1(DE3), colony PCR was used to verify that the plasmid was correct. Target bands (2086 bp) can be observed at the position around 2000 bp (Fig. 1). | After transforming the plasmid into ''E. coli''1(DE3), colony PCR was used to verify that the plasmid was correct. Target bands (2086 bp) can be observed at the position around 2000 bp (Fig. 1). | ||
| − | [[File:T--XMU-China--137.png| | + | [[File:T--XMU-China--137.png|300px]] |
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'''Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195137_pSB1C3.''' | '''Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195137_pSB1C3.''' | ||
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The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by arabinose, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image of PirA-his (Fig. 1), the target protein (14.4 kDa) can be observed at the position between 25 kDa and 14 kDa on the purified protein lanes (FR). | The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by arabinose, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image of PirA-his (Fig. 1), the target protein (14.4 kDa) can be observed at the position between 25 kDa and 14 kDa on the purified protein lanes (FR). | ||
| − | [[File:T--XMU-China--132-sds.png| | + | [[File:T--XMU-China--132-sds.png|300px]] |
'''Fig. 1 SDS-PAGE analysis of protein in lysate of ''E. coli'' BL21(DE3) and the elution samples. '''Target bands (14.4 kDa) can be observed at the position between 25 kDa and 14 kDa. | '''Fig. 1 SDS-PAGE analysis of protein in lysate of ''E. coli'' BL21(DE3) and the elution samples. '''Target bands (14.4 kDa) can be observed at the position between 25 kDa and 14 kDa. | ||
Revision as of 19:30, 10 October 2022
pirA-his
Biology
PirA
PirA (a 111-residue protein) and PirB (a 438-residue protein) consist of the binary photorhabdus insect-related (Pir) toxins PirABvp. These toxins secreted from Vibrio parahaemolyticus cause an emerging disease called acute hepatopancreatic necrosis disease (AHPND) directly in shrimp aquaculture. PirA was reported to facilitate target-specific recognition by binding to certain ligands on the cell membrane/receptor (1).
Usage and design
In order to get PirA and purify it for further test with its receptors, a His-tag (6×His) was fused to the C-terminal of PirA. We used both BBa_I0500 and BBa_B0034 to construct the expression system and obtained the composite part BBa_K4195137, which are assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmid was transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.
To verify that the receptors we chose in our project design can bind to toxins, we constructed this circuit to purify PirA.
Characterization
1. Identification
After transforming the plasmid into E. coli1(DE3), colony PCR was used to verify that the plasmid was correct. Target bands (2086 bp) can be observed at the position around 2000 bp (Fig. 1).
Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195137_pSB1C3.
2. Protein purification of the toxins
The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by arabinose, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image of PirA-his (Fig. 1), the target protein (14.4 kDa) can be observed at the position between 25 kDa and 14 kDa on the purified protein lanes (FR).
Fig. 1 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples. Target bands (14.4 kDa) can be observed at the position between 25 kDa and 14 kDa.
Reference
1. S.-J. Lin, K.-C. Hsu, H.-C. Wang, Structural Insights into the Cytotoxic Mechanism of Vibrio parahaemolyticus PirAvp and PirBvp Toxins. Marine Drugs. 15, 373 (2017).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 136
- 1000COMPATIBLE WITH RFC[1000]