Difference between revisions of "Part:BBa K4195036"

Revision as of 19:53, 10 October 2022

his-pirB

Biology

PirB

PirA (a 111-residue protein) and PirB (a 438-residue protein) consist of the binary photorhabdus insect-related (Pir) toxins PirAB^vp. These toxins from Vibrio parahaemolyticus causes an emerging disease called acute hepatopancreatic necrosis disease (AHPND) directly in shrimp aquaculture. PirB was reported to has the ability to form a pore on the cell membrane that causes cell death (1).

Usage and design

In order to get PirB and further purify it for further test with its receptors, a His-tag (6×His) was fused to the N-terminal of PirB. We used both BBa_I0500 and BBa_B0034 to construct the expression system and obtained the composite part BBa_K4195138, which are assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmids 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 PirB.

Characterization

1. Identification

After transforming the plasmid into E. coli BL21(DE3), colony PCR was used to verify that the plasmid was correct. Target bands (3070 bp) can be observed at the position around 3000 bp (Fig. 1).

Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195138_pSB1C3.

2.Protein purification of the toxins

After the first time of purification, we found that the protein was poorly expressed, so we did not obtain the target protein. Therefore, we constructed this part on the expression vector pET-28a(+) by Gibson assembly, then transformed the plasmid into E. coli BL21(DE3). The positive transformants were selected by kanamycin and confirmed by colony PCR (Fig. 1) and sequencing.

300px

Fig. 2 DNA gel electrophoresis of the colony PCR products of his-PirB_pET-28a(+).Target bands (1539 bp) can be observed at the position between 2000bp and 1500 bp.

However, after the second round of purification, we found that the protein expression remained low as the first time. To improve the efficiency of expression, we co-transformed another plasmid that could express molecular chaperones GroES and GroEL with his-PirB_pET-28a(+) into E. coli BL21(DE3). After being cultivated and induced by IPTG, 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 his-PirB (Fig. 2), the target protein (51.9 kDa) can be observed at the position around 50 kDa on the purified protein lanes (FR).

Fig. 3 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples. Target bands (51.9 kDa) can be observed at the position between 60kDa and 50 kDa.

Reference

1. S.-J. Lin, K.-C. Hsu, H.-C. Wang, Structural Insights into the Cytotoxic Mechanism of Vibrio parahaemolyticus PirA^vp and PirB^vp Toxins. Marine Drugs. 15, 373 (2017).

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
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 136
1000
COMPATIBLE WITH RFC[1000]

@@ Line 13: / Line 13: @@
 ===Usage and design===
-In order to get PirB and further purify it for further test with its receptors, a His-tag (6×His) was fused to the N-terminal of PirB. We used both <partinfo>BBa_I0500</partinfo> and <partinfo>BBa_B0034</partinfo> to construct the expression system and obtained the composite part BBa_K4195138, which are assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmids was transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.
+In order to get PirB and further purify it for further test with its receptors, a His-tag (6×His) was fused to the N-terminal of PirB. We used both <partinfo>BBa_I0500</partinfo> and <partinfo>BBa_B0034</partinfo> to construct the expression system and obtained the composite part <partinfo>BBa_K4195138</partinfo>, which are assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmids 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 PirB.
-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. 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|300px]]
+After transforming the plasmid into E. coli BL21(DE3), colony PCR was used to verify that the plasmid was correct. Target bands (3070 bp) can be observed at the position around 3000 bp (Fig. 1).
+[[File:T--XMU-China--138.png|300px]]
-'''Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195137_pSB1C3.'''
-====2. Protein purification of the toxins====
+'''Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195138_pSB1C3.'''
-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).
+====2.Protein purification of the toxins====
-[[File:T--XMU-China--132-sds.png|300px]]
+After the first time of purification, we found that the protein was poorly expressed, so we did not obtain the target protein. Therefore, we constructed this part on the expression vector pET-28a(+) by Gibson assembly, then transformed the plasmid into ''E. coli'' BL21(DE3). The positive transformants were selected by kanamycin and confirmed by colony PCR (Fig. 1) and sequencing.
+[[File:File:T--XMU-China--036.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. 2 DNA gel electrophoresis of the colony PCR products of his-PirB_pET-28a(+)'''.Target bands (1539 bp) can be observed at the position between 2000bp and 1500 bp.
+However, after the second round of purification, we found that the protein expression remained low as the first time. To improve the efficiency of expression, we co-transformed another plasmid that could express molecular chaperones GroES and GroEL with his-PirB_pET-28a(+) into ''E. coli'' BL21(DE3). After being cultivated and induced by IPTG, 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 his-PirB (Fig. 2), the target protein (51.9 kDa) can be observed at the position around 50 kDa on the purified protein lanes (FR).
+[[File:T--XMU-China--138-sds.png|300px]]
+'''Fig. 3 SDS-PAGE analysis of protein in lysate of ''E. coli'' BL21(DE3) and the elution samples.''' Target bands (51.9 kDa) can be observed at the position between 60kDa and 50 kDa.
 ===Reference===