Part:BBa_K771205
Vio C with Membrane Anchor 5
This part is composed with two parts, the Membrane Anchor 5 and Violacein synthetic pathway enzyme vioC. 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]
Background
Violacein is a violet pigment produced by several bacteria through pathway of 5 related genes, vioA, vioB, vioC, vioD and vioE. It was initially discovered in Chromobacterium violaceum. This metabolite of tryptophan has such special applications as antibacterial, anti-trypanocidal, anti-ulcerogenic, and anticancer drugs. There are three branches in this pathway which could verify the efficiency of the Membrane Rudder.
Violacein synthetic pathway
The violacein biosynthetic pathway includes 5 key enzymes which work in conjunction. VioA, a flavoenzyme and VioB, a heme protein, work together to oxidize and dimerize tryptophen into IPA imine dimer. Then vioE induces a indole rearrangement,producing prodeoxyviolacein acid, also known as PVA.
VioA, VioB and VioE catalyze a sequential reaction to produce PVA. There exists one intrinsic E.coli enzyme that aids in an additional side reaction, further modifying PVA into a green pigment called deoxychromoviridans.
The last two enzymes, VioC and VioD are flavin-dependent oxygenases. VioC alone transforms PVA into a purple pigment called deoxyviolacein, while Vio C could also act cooperatively with VioD. VioD hydroxylates 5-position indole ring, then the other 2-position indole ring is processed by VioC to create the oxindole, and in this way violacein is produced.
Design of Experiment
In Violacein Membrane Rudder System, VioA, VioB, VioE and VioC constitutively aggregate. When blue light is present, VioD with Membrane Anchor 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.
As the figure shows, the upperstream Membrane Anchor part contains ssdsbA(signal peptide), VVD(light sensitive protein), lgt(transmembrane domain), GBD ligand(interaction domain with Membrane Anchor 4), and vioC. The VVD can form dimer with Membrane Anchor 6 under light, which is connected to vioD, 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
- 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 494
Illegal AgeI site found at 1339 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 960
| n/a | Vio C with Membrane Anchor 5 |