Part:BBa_K771206
Vio D with Membrane Anchor 6
This part is composed with two parts, the Membrane Anchor 6 and Violacein synthetic pathway enzyme vioD. The enzyme can be aggregated with other membrane anchors, composing the Membrane Magic system. For more information, please visit [http://2012.igem.org/Team:SJTU-BioX-Shanghai 2012 SJTU-BioX-Shanghai wiki]
Violacein biosynthetic pathway
Violacein is a violet pigment produced by several bacteria through pathway of 5 related genes, vioA, vioB, vioC, vioD and vioE. VioD is one of the key enzyme that function in the production of our main end-product, violacein. The detailed synthetic flux is shown below.
Usage and Biology
In our Violacein Membrane Rudder System, VioA, VioB, VioE and VioC constitutively aggregate. When blue light is present, VioD with Membrane Anchor 6 would aggregate with complex of VioA,B,C and E, making violacein the dominnant final product.But if you turn the light off, VioD with Membrane Anchor would dissociate with VioA,B,C and E. Thus the biosynthetic pathway for deoxyviolacein is switched on.
In on word, part BBa_K771206 determines the violacein producing flux. As the figure of our design shows, the upperstream Membrane Anchor part contains ssdsbA(signal peptide), VVD(light sensitive protein), lgt(transmembrane domain), and vioD. The VVD can form dimer with Membrane Anchor 5 under light, which is connected to vioC, thus stimulating vioA-B-E-C-D pathway, producing violacein
Construct these Membrane anchor fusion proteins into appropriate expression vectors, and induce protein induction under 0.2% L-arabinose. As the Membrane Anchor protein fused with vio genes expresses, the product violacein, deoxyviolacein and a side product deoxychromoviridans could be synthased. Extract products, and run HPLC for quantitative results.
The HPLC results show that the peak of deoxyviolacein appears at about 9 minutes after the injection. We find out that under the induction of light, production of deoxyviolacein was inhibited (almost 0%) while most products were violacein. Without the light induction, however, the pathway leading to the deoxyviolacein was initiated and thus deoxyviolacein was produced(about 8%). Such results confirmed our membrane rudder system and indicated that by controlling the extracellular signal, such as light, we are able to manipulate the branched chain reactions thus producing the target products we desire.
Related Parts
VioA with Membrane Anchor 2 (BBa_K771201)
VioB with Membrane Anchor 3 (BBa_K771202)
VioE with Membrane Anchor 4 (BBa_K771203)
VioC with Membrane Anchor 5 (BBa_K771204)
VioC with Membrane Anchor 5 (BBa_K771205)
VioD with Membrane Anchor 6 (BBa_K771206)
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1465
Illegal XbaI site found at 1477 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1465
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1465
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1465
Illegal XbaI site found at 1477 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1465
Illegal XbaI site found at 1477
Illegal AgeI site found at 494
Illegal AgeI site found at 1339 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 960
Illegal SapI.rc site found at 2458
Illegal SapI.rc site found at 2533
| n/a | Vio D with Membrane Anchor 6 |