Difference between revisions of "Part:BBa K3187012"

Revision as of 12:06, 17 October 2019

pTet sfGFP-P22 Bacteriophage Scaffolding Protein x Coat Protein pT7 (Convergent)

Name: pTeTW3con2-ptet-sfGFP-StrepTag-SP—CP+LPETGG-pT7
Size [bp]: 2932
Size [kDa]: 46.1 (sfGFP – Strep-Tag – Scaffold Protein) + 48.9 (Coat Protein - Strep-Tag - LPETGG)
Origin: Synthetic?
Components: tetA promoter, T7 promoter, T7 terminator, Lac Operator, RBS, Coat Protein, Scaffold Protein, sfGFP, LPETGG, Strep-Tag II
Feature: Production of P22 Virus-like particles with a modifiable surface and sfGFP as cargo

Introduction:

This composite part is a dual expression plasmid with pTeTW3con2 as backbone and two convergent expression sites. The tetA promoter is inducible with anhydrotetracycline (AHT) and the site <a href="https://parts.igem.org/Part:BBa_K3187043"target="_blank">(BBa_K3187043)</a> encodes the fusion protein of sfGFP, Strep-Tag and scaffold protein. The T7 promoter can be induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and the site <a href="https://parts.igem.org/Part:BBa_K3187044"target="_blank">(BBa_K3187044 )</a> encodes the fusion protein of coat protein, Strep-Tag and LPETGG. The composite part can therefore be used for the production of P22 Virus-like particles (VLPs), as they consist of coat and scaffold protein.

The scaffold fusion protein also includes a Strep-Tag for purification, as well as sfGFP. As a result of the fusion of sfGFP with the scaffold protein the VLPs will contain sfGFP as a cargo on the inside.

The coat fusion protein also includes a Strep-Tag <a href="https://parts.igem.org/Part:BBa_K3187025"target="_blank">(BBa_K3187025 )</a> for purification and a LPETGG tag <a href="https://parts.igem.org/Part:BBa_K3187019"target="_blank">(BBa_K3187019 )</a> . As a result of the fusion of the LEPTGG tag with the coat protein the VLPs will present the tag on the outside. This tag is the recognition sequence of Sortase A7M <a href="https://parts.igem.org/Part:BBa_K3187028"target="_blank">(BBa_K3187028 )</a> which catalyzes a covalent bond of the LPETGG and a poly G tag. On this basis the VLPs can be modified on the outside using SortaseA7M after the assembly of the coat and scaffold proteins.

The expression levels of both sites were characterized using the <a href="https://parts.igem.org/Part:BBa_K3187028"target="_blank">BBa_K3187011</a>.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 76
Illegal XbaI site found at 2900
Illegal PstI site found at 1334
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 76
Illegal NheI site found at 1339
Illegal PstI site found at 1334
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 76
Illegal BamHI site found at 796
Illegal XhoI site found at 1499
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 76
Illegal XbaI site found at 2900
Illegal PstI site found at 1334
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 76
Illegal XbaI site found at 2900
Illegal PstI site found at 1334
Illegal NgoMIV site found at 1185
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 94

@@ Line 19: / Line 19: @@
 <p>
      This composite part is a dual expression plasmid with pTeTW3con2 as backbone and two convergent expression sites. The tetA promoter is
-     inducible with anhydrotetracycline (AHT) and the site <a href="https://parts.igem.org/Part:BBa_K3187043" target="_blank">(BBa_K3187043)</a>encodes the fusion protein of sfGFP, Strep-Tag and scaffold
+     inducible with anhydrotetracycline (AHT) and the site <a href="https://parts.igem.org/Part:BBa_K3187043"target="_blank">(BBa_K3187043)</a> encodes the fusion protein of sfGFP, Strep-Tag and scaffold
-     protein. The T7 promoter can be induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and the site<a href="https://parts.igem.org/Part:BBa_K3187044" target="_blank">(BBa_K3187044
+     protein. The T7 promoter can be induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and the site <a href="https://parts.igem.org/Part:BBa_K3187044"target="_blank">(BBa_K3187044
          )</a> encodes the
      fusion protein of coat protein, Strep-Tag and LPETGG. The composite part can therefore be used for the production of
@@ Line 28: / Line 28: @@
 of sfGFP with the scaffold protein the VLPs will contain sfGFP as a cargo on the inside.
 <p>
-     The coat fusion protein also includes a Strep-Tag <a href="https://parts.igem.org/Part:BBa_K3187025" target="_blank">(BBa_K3187025
+     The coat fusion protein also includes a Strep-Tag <a href="https://parts.igem.org/Part:BBa_K3187025"target="_blank">(BBa_K3187025
-         )</a> for purification and a LPETGG tag<a href="https://parts.igem.org/Part:BBa_K3187019" target="_blank">(BBa_K3187019
+         )</a> for purification and a LPETGG tag <a href="https://parts.igem.org/Part:BBa_K3187019"target="_blank">(BBa_K3187019
          )</a> . As a result of the fusion of
      the LEPTGG tag with the coat protein the VLPs will present the tag on the outside. This tag is the recognition
-     sequence of Sortase A7M<a href="https://parts.igem.org/Part:BBa_K3187028" target="_blank">(BBa_K3187028
+     sequence of Sortase A7M <a href="https://parts.igem.org/Part:BBa_K3187028"target="_blank">(BBa_K3187028
          )</a> which catalyzes a covalent bond of the LPETGG and a poly G tag. On this basis the VLPs can
      be
@@ Line 39: / Line 39: @@
 <p>
-     The expression levels of both sites were characterized using the <a href="https://parts.igem.org/Part:BBa_K3187028" target="_blank">BBa_K3187011</a>.
+     The expression levels of both sites were characterized using the <a href="https://parts.igem.org/Part:BBa_K3187028"target="_blank">BBa_K3187011</a>.
 </p>