Difference between revisions of "Part:BBa K3482041"

Latest revision as of 19:40, 27 October 2020

truncated azurin with 3X FLAG

Truncated version of azurin fused with a 3xFLAG tag

Usage and Biology

Azurin is a blue copper-binding redox protein originally found in the periplasmic membrane of Pseudomonas aeruginosa. It has recently been discovered to have an l anticancer effect by targeting the well-known p53 apoptosis pathway and by preferentially entering cancerous cells.

We used this part to test if azurin was produced in our bacteria.

Characterization

To verify the correct expression of the azurin that we successfully cloned into our sponge plasmid and purified, we concatenated a widely used 3XFLAG tag at the C-terminus of our azurin gene (BBa_3482040). This allowed us to perform a Western Blot, which allowed us to confirm the presence or absence of our expressed azurin. In addition, we modified the existing part BBa_K2500001 (truncated azurin uploaded by the ETH Zürich team of 2017) with either the secretion tag pelB or NSP4[4] and investigated its secretion capability. We used an E. coli Nissle 1917 strain, which as been transformed with a modified sponge plasmid (usable in combination with a repressilator plasmid in the future) which contains our target genes constitutively expressed under a pTet promoter (BBa_K3482040, BBa_K3482041, BBa_K3482038, and BBa_K3482037) to performe a Western Blot with overnight cultures set to an OD600 of 2.0. The culture supernatant and lysate (pellet) were analyzed.

Western blot analysis of flag-tagged azurin from 4 different parts under 2 conditions: supernatant or cell lysate. Size range is shown around 15 kDa. Immunoblotting after SDS-PAGE migration, membrane transfer, and analysis performed with 3XFLAG antibody. Samples are as follow: 1 = BBa_K3482040 = azurin-3XFLAG; 2 = BBa_K3482041 = truncated azurin-3XFLAG; 3 = BBa_K3482038 = pelB-5D-truncated azurin-3XFLAG; 4 = BBa_K3482037= NSP4-truncated azurin-3XFLAG.

We can observe the presence of the flag protein in all cell lysate extracts. In addition, we can see that the full-length azurin as well as both versions with a secretion tag (pelB or NSP4)(samples 3 and 4) are present in the supernatant This leads us to conclude that our azurin is indeed expressed and secreted.

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
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 To verify the correct expression of the azurin that we successfully cloned into our sponge plasmid and purified, we concatenated a widely used 3XFLAG tag at the C-terminus of our azurin gene (BBa_3482040). This allowed us to perform a Western Blot, which allowed us to confirm the presence or absence of our expressed azurin. In addition, we modified the existing part BBa_K2500001  (truncated azurin uploaded by the ETH Zürich team of 2017) with either the secretion tag pelB or NSP4[4] and investigated its secretion capability.
-E.coli Nissle 1917, which as been transformed with a modified sponge plasmid (usable in combination with a repressilator plasmid in the future) which contains our target genes constitutively expressed under a pTET promoter (BBa_K3482040, BBa_K3482041, BBa_K3482038, and BBa_K3482037). We then performed a western blot with overnight cultures set to OD600 of 2.0 of culture supernatant and lysate (pellet) were analyzed
+We used an <i>E. coli Nissle 1917</i> strain, which as been transformed with a modified sponge plasmid (usable in combination with a repressilator plasmid in the future) which contains our target genes constitutively expressed under a pTet promoter (BBa_K3482040, BBa_K3482041, BBa_K3482038, and BBa_K3482037) to performe a Western Blot with overnight cultures set to an OD600 of 2.0. The culture supernatant and lysate (pellet) were analyzed.
 [[File:BBa K3482040 azurin purification.png|400px]]
-Western blot analysis of flagged azurin from 4 different parts under 2 conditions: supernatant or cell lysate as “pellet”. Size range is shown around 15 kDa.  Analysis performed by the wet lab team. Immunoblotting after SDS-PAGE migration, membrane transfer, and analysis performed with 3XFLAG antibody.
+Western blot analysis of flag-tagged azurin from 4 different parts under 2 conditions: supernatant or cell lysate. Size range is shown around 15 kDa.  Immunoblotting after SDS-PAGE migration, membrane transfer, and analysis performed with 3XFLAG antibody.
 Samples are as follow: 1 = BBa_K3482040 = azurin-3XFLAG; 2 = BBa_K3482041 = truncated azurin-3XFLAG; 3 = BBa_K3482038 = pelB-5D-truncated azurin-3XFLAG; 4 = BBa_K3482037= NSP4-truncated azurin-3XFLAG.
-We can observe the presence in the immunoblotting of the flag protein in all cell lysate extract. In addition, we can see that both of the truncated versions of azurin which were originally not secreted (samples 3 and 4) are present in the supernatant, if necessary with a secretion tag concatenated. This leads us to conclude that our azurin is indeed expressed and secreted.
+We can observe the presence of the flag protein in all cell lysate extracts. In addition, we can see that the full-length azurin as well as both versions with a secretion tag (pelB or NSP4)(samples 3 and 4) are present in the supernatant This leads us to conclude that our azurin is indeed expressed and secreted.