Difference between revisions of "Part:BBa K771206"

Revision as of 05:53, 28 September 2012 (view source) Zhiantinglan (Talk | contribs) (→‎Violacein biosynthetic pathway) ← Older edit		Revision as of 10:54, 2 October 2012 (view source) Albertwyq (Talk | contribs) (→‎Violacein biosynthetic pathway) Newer edit →
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	For more information, please visit [http://2012.igem.org/Team:SJTU-BioX-Shanghai  2012 SJTU-BioX-Shanghai wiki]		For more information, please visit [http://2012.igem.org/Team:SJTU-BioX-Shanghai  2012 SJTU-BioX-Shanghai wiki]
−	===Violacein biosynthetic pathway===	+	==Background==
−	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.	+	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.
−	[[Image:12SJTU VioPathway.png|350px|center|The violacein biosynthetic pathway]]	+	===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.
		+
		+	[[Image:12SJTU_VioPathway.png|400px|center|The violacein biosynthetic pathway]]
		+
		+	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==
		+
		+	===Switch of reaction direction===
		+
		+	We constructed our device as demonstrated in Fig.2. 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.
		+
		+	[[Image:12SJTU-VIO.png|center|400px|thumb]]
		+
		+	For VioB and VioE only function normally in dimereic state, free VioB and free VioE were coexpressed with membrane assembly carrying VioA,B,C,D and E to ensure the normal function of the whole system.
		+
		+	To characterize the practical effect of Violacein Membrane Rudder System, we coexpressed all proteins in Fig.2 with free VioB and VioE. Bacteria in experimental group were induced at a L-Arabinose concentration of 0.1% under blue light while control group were induced in the dark with other conditions all the same.
		+
		+
		+	===Suppression of side-product===
		+
		+	According to the mechanism behind our design of membrane rudder, a considerable reduction of deoxychromoviridans production is expected. Under light signal, VioD and VioC get close to other Vio enzymes. The assembly of sequential enzymes helps substrate flow to downstream enzymes, making violacein more preferably synthesized . The overall amount of available tryptophan in a bacterium is fixed, so preference to the production of violacein would inevitably cut the supply to the other branches of reaction.
		+
		+	To verify our assumption, free VioABCDE genes are also transformed into E.coli as control group.
		+
		+	==Results and Discussion==
		+
		+	===Overview===
		+
		+	By attaching the core enzymes to the VIVID, we managed to control the flow of branched chain reaction, such as violacein biosynthetic pathway. Results indicate that the extracts of bacterial cultures induced by light contained almost no deoxyviolacein while it was produced in the samples restrained from light. Moreover, a significant decrease of side products such as deoxychomoviridans was detected when membrane complex system was present.
		+
		+	===Switch Analysis===
		+	[[Image:12SJTU VioHPLC.jpg|500px|thumb|right|HPLC result of culture extraction]]The switch of the reaction can be realized by the extracellular signals. Through the light induction, the reaction producing deoxyviolacein by VioC alone is inhibited due to the lack of interaction with PVA. On the other hand, as long as light is restrained from the bacteria, however, the above reaction is initiated leading to the production of deoxyviolacein.
		+
		+	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.
		+
		+	===Suppression of side reaction===
		+	[[Image:12sjtu-green.png|thumb|250px|left|''Fig4'':The HPLC peak area of deoxychromoviridans from sample with light induction and free vioABCDE control group.]]
		+	<br\>
		+
		+	Membrane complex system would help to reduce the amount of side-products to some extent. Under the case that the enzymes involved in the side reaction are situated in the cytoplasm, they are less competent than the core enzymes attached on the membrane to mediate the subsequent reactions due to the spatial obstacle.
		+
		+	From the HPLC results, we detected a distinct decrease of side-products deoxychomoviridans. Judging from the peak area, we find out that the amount of deoxychromoviridans from rbs-vio control group is nearly eight times that from light-induced group. Such experiment results coincided with our anticipation and demonstrated that the membrane complex system would enhance the usage of substrates and thus reduce the amount of intermediate products and other subsequent side-products.
		+
		+	<br\><br\><br\><br\><br\>
		+
		+	==Violacein Protocols==
		+
		+	===Induction and Extraction===
		+	**Incubate the monoclonal from medium in a shaker at 37°C overnight
		+	**Add 0.1% Arabinose, 1mM Ferrous ammonium sulfate and 40 μM to start induction for another 6 hours
		+	**Centrifuge for 1 min at 10000rpm, remove the LB Medium
		+	**Add 800 μL 10%SDS and break cells using ultrasound
		+	**Add 800 μL ethyl acetate to extract violacein/deoxyviolacein
		+	**Use rotovap to concentrate violacein and resuspend with methanol
		+	===HPLC===
		+	**Column: C18, 15cm
		+	**Mobile phase: 75% methanol, 25% ddH2O
		+	**Injection volume: 20 μL
		+	**Speed: 0.5 mL/min
		+	**Detection: 230 nm
		+
		+	==HPLC Data==
		+	[[Image:12SJTU_HPLC_VioABCDE_Nolight.png|thumb|800px|center|HPLC_VioABCDE_Nolight]]
		+
		+	[[Image:12SJTU_HPLC_VioABCDE_Light.png|thumb|800px|center|HPLC_VioABCDE_Light]]
		+
		+
		+
		+	===Related Parts===
		+	[https://parts.igem.org/wiki/index.php?title=Part:BBa_K771201 VioA with Membrane Anchor 2 (BBa_K771201)]
		+
		+	[https://parts.igem.org/wiki/index.php?title=Part:BBa_K771202 VioB with Membrane Anchor 3 (BBa_K771202)]
		+
		+	[https://parts.igem.org/wiki/index.php?title=Part:BBa_K771203 VioE with Membrane Anchor 4 (BBa_K771203)]
		+
		+	[https://parts.igem.org/wiki/index.php?title=Part:BBa_K771204 VioC with Membrane Anchor 5 (BBa_K771204)]
		+
		+	[https://parts.igem.org/wiki/index.php?title=Part:BBa_K771205 VIoC with Membrane Anchor 5 (BBa_K771205)]
		+
		+	[https://parts.igem.org/wiki/index.php?title=Part:BBa_K771206 Viod with Membrane Anchor 6 (BBa_K771206)]
	===Usage and Biology===		===Usage and Biology===

---

Revision as of 10:54, 2 October 2012

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]

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

Switch of reaction direction

We constructed our device as demonstrated in Fig.2. 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.

For VioB and VioE only function normally in dimereic state, free VioB and free VioE were coexpressed with membrane assembly carrying VioA,B,C,D and E to ensure the normal function of the whole system.

To characterize the practical effect of Violacein Membrane Rudder System, we coexpressed all proteins in Fig.2 with free VioB and VioE. Bacteria in experimental group were induced at a L-Arabinose concentration of 0.1% under blue light while control group were induced in the dark with other conditions all the same.

Suppression of side-product

According to the mechanism behind our design of membrane rudder, a considerable reduction of deoxychromoviridans production is expected. Under light signal, VioD and VioC get close to other Vio enzymes. The assembly of sequential enzymes helps substrate flow to downstream enzymes, making violacein more preferably synthesized . The overall amount of available tryptophan in a bacterium is fixed, so preference to the production of violacein would inevitably cut the supply to the other branches of reaction.

To verify our assumption, free VioABCDE genes are also transformed into E.coli as control group.

Results and Discussion

Overview

By attaching the core enzymes to the VIVID, we managed to control the flow of branched chain reaction, such as violacein biosynthetic pathway. Results indicate that the extracts of bacterial cultures induced by light contained almost no deoxyviolacein while it was produced in the samples restrained from light. Moreover, a significant decrease of side products such as deoxychomoviridans was detected when membrane complex system was present.

Switch Analysis

The switch of the reaction can be realized by the extracellular signals. Through the light induction, the reaction producing deoxyviolacein by VioC alone is inhibited due to the lack of interaction with PVA. On the other hand, as long as light is restrained from the bacteria, however, the above reaction is initiated leading to the production of deoxyviolacein.

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.

Suppression of side reaction

<br\>

Membrane complex system would help to reduce the amount of side-products to some extent. Under the case that the enzymes involved in the side reaction are situated in the cytoplasm, they are less competent than the core enzymes attached on the membrane to mediate the subsequent reactions due to the spatial obstacle.

From the HPLC results, we detected a distinct decrease of side-products deoxychomoviridans. Judging from the peak area, we find out that the amount of deoxychromoviridans from rbs-vio control group is nearly eight times that from light-induced group. Such experiment results coincided with our anticipation and demonstrated that the membrane complex system would enhance the usage of substrates and thus reduce the amount of intermediate products and other subsequent side-products.

<br\><br\><br\><br\><br\>

Violacein Protocols

Induction and Extraction

• • Incubate the monoclonal from medium in a shaker at 37°C overnight
  • Add 0.1% Arabinose, 1mM Ferrous ammonium sulfate and 40 μM to start induction for another 6 hours
  • Centrifuge for 1 min at 10000rpm, remove the LB Medium
  • Add 800 μL 10%SDS and break cells using ultrasound
  • Add 800 μL ethyl acetate to extract violacein/deoxyviolacein
  • Use rotovap to concentrate violacein and resuspend with methanol

HPLC

• • Column: C18, 15cm
  • Mobile phase: 75% methanol, 25% ddH2O
  • Injection volume: 20 μL
  • Speed: 0.5 mL/min
  • Detection: 230 nm

HPLC Data

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)

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