Team:SJTU-BioX-Shanghai/Project/project2.1

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=Biosynthesis - Violacein=
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=Membrane Rudder <br><br> - Violacein & Deoxyviolacein Synthetic Pathway=
{{Template:12SJTU_part_summary_head}}
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*'''State of the art'''
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Violacein is a purple chromobacterial pigment produced from tryptophan. In its biosynthetic pathway there are 3 possible branches and 2 side products. The five key enzymes of ''Chromobacterium violaceum'' could be expressed in ''E.Coli'' and corresponding parts are available in the Registry. The Cambridge  2009 iGEM team had succesfully achieved the production of violacein.
+
*'''State of the art'''  
-
*'''Aim'''
+
Dynamically and artificially regulating the direction of biochemical pathway ''in vivo'' has remained a challenge for scientists. We are now to achieve this goal through controlling the aggregation state of different enzymes in branched biological reactions based on Membrane Scaffold. We named this universal device ''Membrane Rudder''.
-
Our membrane rudder calls for a branched reaction to test its efficiency. We wanted to examine the advantages of membrane system in adjusting quantity of different products.
+
 +
Violacein is a purple chromobacterial pigment synthesized from the basic amino acid tryptophan. Its biosynthetic pathway contains 3 branches and produces 3 final products, which are violacein, deoxyviolacein and deoxychromoviridans respectively. Thus it is incorporated to test the feasibility of ''Membrane Rudder''
 +
 
 +
 
 +
*'''Aims'''
 +
To switch the direction of Violacein and deoxyviolacein synthetic pathway through light signal
 +
 
 +
To switch the direction of Violacein and deoxyviolacein synthetic pathway through RNA signal
 +
 
 +
To justify the universality of ''Membrane Rudder''
 +
 
 +
 
*'''Achievements'''
*'''Achievements'''
-
We modified the five vio parts and inserted them in the membrane assembly, producing a plasmid with violacein biosynthetic enzyme, light-inducing protein as well as a membrane protein. Analysis of bacteria extracts showed a satisfying decrease in side products between light-induced samples and the control group. These results demonstrate a rather promising way of using membrane rudder to switch the direction of reaction.  
+
 
 +
''Direction switched'' successfully in Violacein synthetic pathway by light signal
 +
 
 +
''8-fold yields decrease'' of side product deoxychromaviridans with ''Membrane Rudder'' compared to group with free cytoplasmic enzyme
 +
 
 +
''A novel device constructed'' that connects post-translational Membrane Scaffold system to genetic circuits by recruiting RNA as a controlling signal.
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==Background==
==Background==
-
Violacein is a 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.
+
Dynamically and artificially regulating the direction of biochemical pathway ''in vivo'' has remained a challenge for scientists. Based on Membrane Scaffold, we are now trying to achieve this goal through controlling the aggregation state of different enzymes in branched biological reactions. We named this universal device ''Membrane Rudder''. Theoretically this device could sense a large amount of signals. We chose blue light and rationally designed RNA molecule D0 as controlling signal. Branched violacein & deoxyviolacein synthetic pathway is recruited to verify the feasibility of ''Membrane Rudder''.
-
There are two interesting branches in this pathway which could be utilized in service of verification of the ''membrane rudder''. We can identify the different end-products by means of separation, such as thin-layer chromatography (TLC), or high-performance liquid chromatography (HPLC). The product proportion change demonstrates the efficiency of alteration in reaction direction.
+
There are three branches in violacein & deoxyviolacein synthetic pathway. We could identify amount of final products through high-performance liquid chromatography (HPLC). The change of products proportion will demonstrate the efficiency of direction alteration.
-
===Violacein Biosynthetic Pathway===
 
-
[[Image:12SJTU_VioPathway.png|thumb|250px|right|''Fig.1'' :The violacein biosynthetic pathway. The purple line indicates the biosynthetic flux of pigment deoxyviolacein and violet. The green line indicates the the biosynthetic flux of pigment deoxychromoviridans.]]
 
-
The violacein biosynthetic pathway includes 5 key enzymes which work in conjunction. VioA, a flavoenzyme and VioB, a heme protein, they work together to oxidize and dimerize tryptophen into IPA imine dimer. Then vioE induces a indole rearrangement,producing prodeoxyviolacein, also known as PVA.
+
===Violacein & Deoxyviolacein Biosynthetic Pathway===
-
One key prerequisite in the metabolic flux is that: Automatically VioA, VioB and VioE can assemble and function 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(''1st main product'').  
+
::[[Image:Figure1-SJTU violacein-pathway.png|thumb|600px|center|''Fig.1:'' :Details of violacein & deoxyviolacein biosynthetic pathway. The left pathway painted in purple indicates the synthetic pathway of purple pigment deoxyviolacein.The right pathway painted in violet indicates the synthetic pathway of purple pigment violacein. The green pathway indicates the nonenzymatic production of green pigment deoxychromoviridans.]]
-
The last two proteins, VioC and VioD are flavin-dependent oxygenases. VioC alone transfers PVA into a purple pigment named deoxyviolacein(''2nd main product''), while vio C could also act sequentially with vioD.
+
The violacein & deoxyviolacein biosynthetic pathway includes 5 key enzymes which work in sequence: Vio A, B, C, D and E. VioA, a flavoenzyme and VioB, a heme protein, work together to oxidize and dimerize tryptophen into IPA imine dimer. Then VioE induces an indole rearrangement, producing prodeoxyviolacein acid, also known as PVA. In a word, enzyme VioA, VioB and VioE convert tryptophen into PVA. Then this biosynthetic pathway is about to branch.
-
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(''3rd main product'') is produced.
+
-
==Design of Membrane Assembly==
+
In ''E.coli'', an additional side reaction could occur, and PVA is converted into a green pigment called deoxychromoviridans.
-
Theoretically our membrane rudder offers a method to fine-tune reaction direction, and in the meantime decrease side-products quantity.
+
-
===Control over reaction flow===
+
The last two enzymes, VioC and VioD are flavin-dependent oxygenases. VioC alone transforms PVA into a purple pigment called deoxyviolacein. VioC could also act cooperatively with VioD. VioD hydroxylates 5-position indole ring, and then the other 2-position indole ring is processed by VioC to create the oxindole. In this way, violacein is produced.
-
Since VioA,VioB and VioE normally aggregate to finish the transfer from tryptophen to PVA, the key to direction of the rest part of pathway is if PVA could be passed on to vioD timely. We intergrated M1-vioA with vioC, and M3-vioE with vioD to assure the PVA would be catalyzed by the next enzyme right away.Through connecting VIVID split protein and VioC and VioD seperately, a light signal could efficiently shorten distance between these two enzymes. As long as VVD is photo-induced and dimerized, they could act like magnets that pull vioC and vioD together, facilitating the indole ring rearrangement under catalization of VioC and VioD,in this case violacein should be the dominant product we expect. Since vioB and vioE normally function in dimer state, pRSFduet with rbs-vioB and rbs-vioE was transformed into ''E.Coli'' to ensure dimerization, then all five functional enzymes could be expressed now.
+
-
To quantify the practical effect of photo-inducing direction alteration, we set a control group and conducted experiments as designed.There was no other difference but one that the control group was induced under absolutely no light signal.
+
-
figure2//膜蛋白图片
+
==Design of Experiment==
-
===Suppression over side-reactions===
+
We tried to create ''Membrane Rudder'' controlled by light signal and RNA signal to switch direction of biochemical reaction dynamically and artificially in ''E.coli''.
-
According to the mechanism behind our design of membrane rudder, a considerable reduction of side-product is expected. Under light signal, VioD and VioC get close to other Vio proteins,the assembly of key enzymes near the membrane also lessens the proportion of dissociative intermediates and makes them easier to be captured by VioD and VioC. The overall amount of available tryptophen in a bacterium is fixed, preferance to the production of violacein would inevitably cut the supply to the other branches of reaction.  
+
===Membrane Rudder Sensing Light Signal===
-
In a word, productivity of side-products will be decreased and tendancy of low production is possible.
+
Light is an intriguing signal to regulate E.coli activity because it is easy to obtain, highly tunable and nontoxic. A metabolite-coupled light-switchable system could be quite fascinating.
-
To verify our assumption, rbs-vio genes were also inserted in the three modified plasmids and transformed into ''E.Coli''.
+
We constructed our device as demonstrated in ''Fig.2''. In light-sensing ''Membrane Rudder'', VioA, VioB, VioE and VioC with interacting Membrane Anchors constitutively aggregate. [https://2012.igem.org/Team:SJTU-BioX-Shanghai/Project/project1.2 VVD], a photoreceptor of ''Neurospora crassa'', can form dimer in the presence of blue light and disassociate as light is off. When blue light is present, VioD with VVD-coupled Membrane Anchor would aggregate with assembly of VioA, B, C and E, making violacein the dominant final product. But when blue light is off, VioD with VVD coupled Membrane Anchor will disassociate with VioA, B, C and E. Thus the biosynthetic pathway for deoxyviolacein is switched on.
 +
 
 +
[[File:12SJTU_VVD_Construction.png|center|500px|thumb|''Fig.2'' :Construction details of light-sensing ''Membrane Rudder''. VVD acts as a blue light sensor.]]
 +
 
 +
For VioB and VioE only function normally in dimeric state, free VioB and free VioE were coexpressed with membrane anchored VioA, B, C, D and E (''Fig.2'')to ensure the normal function of the whole system.
 +
 
 +
Bacteria in experimental group were induced at a L-Arabinose concentration of 0.1%. One group of bacteria expressing full set of light-sensing ''Membrane Rudder'' is incubated under blue light. The other group of bacteria expressing full set of light-sensing ''Membrane Rudder'' are incubated in the dark. In each comparative group, bacteria prepared for light induction and dark incubation are taken from the same sample.
 +
 
 +
The BioBrick Part VVD-MA5-vioC and VVD-MA6-vioD is [http://partsregistry.org/Part:BBa_K771205 Part:BBa_K771205] and [http://partsregistry.org/Part:BBa_K771206 Part:BBa_K771206], respectively. For MA2-VioA, MA3-VioB, MA4-VioE, the corresponding part is [http://partsregistry.org/Part:BBa_K771201 Part:BBa_K771201], [http://partsregistry.org/Part:BBa_K771202 Part:BBa_K771202], [http://partsregistry.org/Part:BBa_K771203 Part:BBa_K771203], respectively.
 +
 
 +
===Membrane Rudder Sensing RNA Signal===
 +
 
 +
Membrane Accelerator and Membrane Rudder are both post-translational controlling device to regulate metabolic flux of the host cell. To connect this relatively isolated post-translational control system to genetic circuits, we employed RNA signal, which is present in cytoplasm. Rationally designed RNA D0 with MS2 and PP7 aptamer domain is recruited. When RNA molecule with this two aptamer domains is present in cells, their cognate aptamer binding proteins can thus aggregate together. Furthermore, if we place RNA D0 (with PP7 and MS2 aptamer domains) under various promoters regulated by different signals, approaches to induce dimerization would be expanded sharply. Thus, Membrane Rudder could sense much more signals.
 +
 
 +
We constructed our device as demonstrated in ''Fig.3''. In RNA-sensing ''Membrane Rudder'', VioA, VioB, VioE and VioC with interacting Membrane Anchors constitutively aggregate. [https://2012.igem.org/Team:SJTU-BioX-Shanghai/Project/project1.2 RNA D0], can aggregate with RNA aptamer binding protein MS2 and PP7. So when RNA D0 is present, VioD with MS2-coupled Membrane Anchor would aggregate with assembly of VioA, B, C and E, making violacein the dominant final product. But when RNA D0 is absent, VioD with MS2-coupled Membrane Anchor will disassociate with VioA, B, C and E. Thus the biosynthetic pathway for deoxyviolacein is switched on.
 +
 
 +
[[File:12SJTU_RNA_construction.png|center|500px|thumb|''Fig.3'' :Construction details of RNA-sensing ''Membrane Rudder''. RNA aptamer binding protein MS2 and PP7 act as an RNA signal sensors.]]
 +
 
 +
Free VioB and free VioE were coexpressed with membrane anchored VioA, B, C, D and E to ensure the normal function of the whole system.
 +
 
 +
Bacteria in experimental group were induced at a L-Arabinose concentration of 0.1%. One group of bacteria expressing full set of RNA-sensing ''Membrane Rudder'' are incubated under blue light. The other group of bacteria expressing full set of RNA-sensing ''Membrane Rudder'' are incubated in the dark. In each comparative group, bacteria prepared for light induction and dark incubation are taken from the same sample.
==Results and Discussion==
==Results and Discussion==
-
===Overview===
+
===Membrane Rudder Sensing Light Signal===
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By attaching the core enzymes to the VIVID, we succeeded to control the flow of branched chain reaction, such as violacein biosynthetic pathway. Results indicate that deoxybiolacein was almost absent from the extracts of bacterial cultures induced by light while samples restrained from light containd deoxyviolacein.(Figure X, compare X and X) Moreover, a significant decrease of side products such as deoxychomoviridans was detected, when membrane complex system was present. (Figure X, compare lane X and X)
+
-
===Switch Analysis===
+
'''Overview'''
-
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.
+
-
[[Image:12SJTU_violacein_production_ratio.JPG|thumb|320px|left|''Fig.2''blablablabla]]
+
By attaching Enzymes VioA, B, C, D and E to Membrane Anchors as shown in ''Fig.2'', we managed to change the proportion of final products by switching on or off blue light signal. When blue light is on, VioA, B, C, D and E aggregate together, making violacein the dominant final product. When blue light is off, VioD with Membrane Anchor will disassociate with VioA, B, C and E. Thus the biosynthetic pathway for deoxyviolacein is switched on.
-
From the HPLC, we find out that under the induction of light, production of deoxyviolacein was inhibited (less than 1%) while most products were violacein. Without the light induction, however, the pathway leading to the deoxyviolacein was initiated and some deoxyviolacein was produced(about 8%). Such results confirmed our membrane rudder system and indicate 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.
+
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===Inhibition of side reaction===
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Moreover, a significant decrease of side products such as deoxychomoviridans was observed when ''Membrane Rudder'' system was present.
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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.
 
-
[[Image:12SJTU_Violacein_production3_Ratio.JPG|thumb|320px|right|''Fig.3''blablablabla]]
 
-
From the HPLC results, we detected a distinct decrease of side-products deoxychomoviridans. Such experiment results coincides with our anticipation and demonstrated the significance membrane complex system has in the reduction of side-products.
+
'''Direction Alteration'''
 +
 
 +
Altering direction of violacein & deoxyviolacein synthetic pathway can be realized by dynamically controlling the aggregation state of crucial enzymes. Through the blue light induction, all five enzymes aggregate together. Thus branch pathway producing deoxyviolacein by VioA, B, E and C is inhibited because PVA is more preferably hydroxylated by VioD. On the other hand, as long as blue light is absent from the bacteria, VioD will disassociate with assembling VioA, B, E and C, leading to the production of deoxyviolacein.
 +
 
 +
[[Image:P1000998.JPG|thumb|400px|center|''Fig.4'' :Collections of different peaks in HPLC results, ready to undergo MS for molecule construction verification.]]
 +
 
 +
We ran an HPLC (SHIMADZU LC-20AP, C18 reversed column) test with the purple samples extracted from the bacteria culture. Then we ran mass spectrometry (Thermo Ultra GC-ISQ) test and thus further confirmed the molecular constitution of main peaks in HPLC results.
 +
 
 +
The HPLC results perfectly fit our prediction.
 +
 
 +
[[Image:12SJTU_vio_propotion4.jpg|thumb|700px|center|''Fig.5'' :HPLC result (right) and the ratio(left) of deoxyviolacein in sample group with blue light induction and group without blue light induction. Red column stands for yield of deoxyviolacein; Blue column stands for yield of violacein.  Fig. B shows the HPLC result from sample without blue light induction. Fig. C shows the HPLC result from sample with from sample under blue light induction.The HPLC results show that the peak of deoxyviolacein appears at about 9 minutes after the injection. The peak of deoxyviolacein is identified according to previously published research. Results indicate that the amount of the deoxyviolacein from the sample without blue light induction is tremendously larger than that from light induced sample]]
 +
 
 +
 
 +
Under the induction of blue light, production of deoxyviolacein was inhibited (to almost 0%) while most products were violacein, which is predicted above. Without blue light induction, however, the pathway leading to the production of deoxyviolacein was turned on and thus considerable amount of deoxyviolacein was produced. All results confirmed the feasibility of our light-sensing ''Membrane Rudder''. Besides, it indicated that by replacing signal sensors and functioning enzymes, we could regulate direction of various branched reactions through diversified signals.
 +
<br>
 +
 
 +
'''Decrease in Side Products'''
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 +
We also found membrane anchored enzymes could help to reduce the amount of side-product(deoxychromaviridans) by 8 fold compared to diffusing cytoplasmic enzymes. This is because rationally designed enzyme assembly facilitate and improve the metabolic flux of biosynthesis. Intermediate PVA is more preferably processed by adjacent membrane anchored VioD or VioC rather than converted into deoxychromaviridans. Actually this is one of the advantages of Membrane Scaffold.
 +
 
 +
[[Image:12SJTU_Virdrans_before_after.jpg|thumb|400px|center|''Fig.5'' :The HPLC peak area of deoxychromoviridans from sample with free VioABCDE(denoted as ''Free'')  and membrane anchored VioABCDE(denoted as Complex). Construction detail of membrane anchored VioABCDE is shown in ''Fig.2''. Bacteria are incubated under light for 6 hours in ''Complex'' group.]]
==Reference==
==Reference==
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3. Shrode, L. B., Z. A. Lewis, et al. (2001). "vvd is required for light adaptation of conidiation-specific genes of Neurospora crassa, but not circadian conidiation." Fungal Genet Biol 32(3): 169-81.
3. Shrode, L. B., Z. A. Lewis, et al. (2001). "vvd is required for light adaptation of conidiation-specific genes of Neurospora crassa, but not circadian conidiation." Fungal Genet Biol 32(3): 169-81.
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 +
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{{Template:12SJTU_footer}}
{{Template:12SJTU_footer}}

Latest revision as of 03:55, 27 October 2012

Membrane Rudder

- Violacein & Deoxyviolacein Synthetic Pathway

  • State of the art

Dynamically and artificially regulating the direction of biochemical pathway in vivo has remained a challenge for scientists. We are now to achieve this goal through controlling the aggregation state of different enzymes in branched biological reactions based on Membrane Scaffold. We named this universal device Membrane Rudder.

Violacein is a purple chromobacterial pigment synthesized from the basic amino acid tryptophan. Its biosynthetic pathway contains 3 branches and produces 3 final products, which are violacein, deoxyviolacein and deoxychromoviridans respectively. Thus it is incorporated to test the feasibility of Membrane Rudder


  • Aims

To switch the direction of Violacein and deoxyviolacein synthetic pathway through light signal

To switch the direction of Violacein and deoxyviolacein synthetic pathway through RNA signal

To justify the universality of Membrane Rudder


  • Achievements

Direction switched successfully in Violacein synthetic pathway by light signal

8-fold yields decrease of side product deoxychromaviridans with Membrane Rudder compared to group with free cytoplasmic enzyme

A novel device constructed that connects post-translational Membrane Scaffold system to genetic circuits by recruiting RNA as a controlling signal.


Background

Dynamically and artificially regulating the direction of biochemical pathway in vivo has remained a challenge for scientists. Based on Membrane Scaffold, we are now trying to achieve this goal through controlling the aggregation state of different enzymes in branched biological reactions. We named this universal device Membrane Rudder. Theoretically this device could sense a large amount of signals. We chose blue light and rationally designed RNA molecule D0 as controlling signal. Branched violacein & deoxyviolacein synthetic pathway is recruited to verify the feasibility of Membrane Rudder.

There are three branches in violacein & deoxyviolacein synthetic pathway. We could identify amount of final products through high-performance liquid chromatography (HPLC). The change of products proportion will demonstrate the efficiency of direction alteration.


Violacein & Deoxyviolacein Biosynthetic Pathway

Fig.1: :Details of violacein & deoxyviolacein biosynthetic pathway. The left pathway painted in purple indicates the synthetic pathway of purple pigment deoxyviolacein.The right pathway painted in violet indicates the synthetic pathway of purple pigment violacein. The green pathway indicates the nonenzymatic production of green pigment deoxychromoviridans.

The violacein & deoxyviolacein biosynthetic pathway includes 5 key enzymes which work in sequence: Vio A, B, C, D and E. VioA, a flavoenzyme and VioB, a heme protein, work together to oxidize and dimerize tryptophen into IPA imine dimer. Then VioE induces an indole rearrangement, producing prodeoxyviolacein acid, also known as PVA. In a word, enzyme VioA, VioB and VioE convert tryptophen into PVA. Then this biosynthetic pathway is about to branch.

In E.coli, an additional side reaction could occur, and PVA is converted 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. VioC could also act cooperatively with VioD. VioD hydroxylates 5-position indole ring, and then the other 2-position indole ring is processed by VioC to create the oxindole. In this way, violacein is produced.

Design of Experiment

We tried to create Membrane Rudder controlled by light signal and RNA signal to switch direction of biochemical reaction dynamically and artificially in E.coli.

Membrane Rudder Sensing Light Signal

Light is an intriguing signal to regulate E.coli activity because it is easy to obtain, highly tunable and nontoxic. A metabolite-coupled light-switchable system could be quite fascinating.

We constructed our device as demonstrated in Fig.2. In light-sensing Membrane Rudder, VioA, VioB, VioE and VioC with interacting Membrane Anchors constitutively aggregate. VVD, a photoreceptor of Neurospora crassa, can form dimer in the presence of blue light and disassociate as light is off. When blue light is present, VioD with VVD-coupled Membrane Anchor would aggregate with assembly of VioA, B, C and E, making violacein the dominant final product. But when blue light is off, VioD with VVD coupled Membrane Anchor will disassociate with VioA, B, C and E. Thus the biosynthetic pathway for deoxyviolacein is switched on.

Fig.2 :Construction details of light-sensing Membrane Rudder. VVD acts as a blue light sensor.

For VioB and VioE only function normally in dimeric state, free VioB and free VioE were coexpressed with membrane anchored VioA, B, C, D and E (Fig.2)to ensure the normal function of the whole system.

Bacteria in experimental group were induced at a L-Arabinose concentration of 0.1%. One group of bacteria expressing full set of light-sensing Membrane Rudder is incubated under blue light. The other group of bacteria expressing full set of light-sensing Membrane Rudder are incubated in the dark. In each comparative group, bacteria prepared for light induction and dark incubation are taken from the same sample.

The BioBrick Part VVD-MA5-vioC and VVD-MA6-vioD is [http://partsregistry.org/Part:BBa_K771205 Part:BBa_K771205] and [http://partsregistry.org/Part:BBa_K771206 Part:BBa_K771206], respectively. For MA2-VioA, MA3-VioB, MA4-VioE, the corresponding part is [http://partsregistry.org/Part:BBa_K771201 Part:BBa_K771201], [http://partsregistry.org/Part:BBa_K771202 Part:BBa_K771202], [http://partsregistry.org/Part:BBa_K771203 Part:BBa_K771203], respectively.

Membrane Rudder Sensing RNA Signal

Membrane Accelerator and Membrane Rudder are both post-translational controlling device to regulate metabolic flux of the host cell. To connect this relatively isolated post-translational control system to genetic circuits, we employed RNA signal, which is present in cytoplasm. Rationally designed RNA D0 with MS2 and PP7 aptamer domain is recruited. When RNA molecule with this two aptamer domains is present in cells, their cognate aptamer binding proteins can thus aggregate together. Furthermore, if we place RNA D0 (with PP7 and MS2 aptamer domains) under various promoters regulated by different signals, approaches to induce dimerization would be expanded sharply. Thus, Membrane Rudder could sense much more signals.

We constructed our device as demonstrated in Fig.3. In RNA-sensing Membrane Rudder, VioA, VioB, VioE and VioC with interacting Membrane Anchors constitutively aggregate. RNA D0, can aggregate with RNA aptamer binding protein MS2 and PP7. So when RNA D0 is present, VioD with MS2-coupled Membrane Anchor would aggregate with assembly of VioA, B, C and E, making violacein the dominant final product. But when RNA D0 is absent, VioD with MS2-coupled Membrane Anchor will disassociate with VioA, B, C and E. Thus the biosynthetic pathway for deoxyviolacein is switched on.

Fig.3 :Construction details of RNA-sensing Membrane Rudder. RNA aptamer binding protein MS2 and PP7 act as an RNA signal sensors.

Free VioB and free VioE were coexpressed with membrane anchored VioA, B, C, D and E to ensure the normal function of the whole system.

Bacteria in experimental group were induced at a L-Arabinose concentration of 0.1%. One group of bacteria expressing full set of RNA-sensing Membrane Rudder are incubated under blue light. The other group of bacteria expressing full set of RNA-sensing Membrane Rudder are incubated in the dark. In each comparative group, bacteria prepared for light induction and dark incubation are taken from the same sample.

Results and Discussion

Membrane Rudder Sensing Light Signal

Overview

By attaching Enzymes VioA, B, C, D and E to Membrane Anchors as shown in Fig.2, we managed to change the proportion of final products by switching on or off blue light signal. When blue light is on, VioA, B, C, D and E aggregate together, making violacein the dominant final product. When blue light is off, VioD with Membrane Anchor will disassociate with VioA, B, C and E. Thus the biosynthetic pathway for deoxyviolacein is switched on.

Moreover, a significant decrease of side products such as deoxychomoviridans was observed when Membrane Rudder system was present.


Direction Alteration

Altering direction of violacein & deoxyviolacein synthetic pathway can be realized by dynamically controlling the aggregation state of crucial enzymes. Through the blue light induction, all five enzymes aggregate together. Thus branch pathway producing deoxyviolacein by VioA, B, E and C is inhibited because PVA is more preferably hydroxylated by VioD. On the other hand, as long as blue light is absent from the bacteria, VioD will disassociate with assembling VioA, B, E and C, leading to the production of deoxyviolacein.

Fig.4 :Collections of different peaks in HPLC results, ready to undergo MS for molecule construction verification.

We ran an HPLC (SHIMADZU LC-20AP, C18 reversed column) test with the purple samples extracted from the bacteria culture. Then we ran mass spectrometry (Thermo Ultra GC-ISQ) test and thus further confirmed the molecular constitution of main peaks in HPLC results.

The HPLC results perfectly fit our prediction.

Fig.5 :HPLC result (right) and the ratio(left) of deoxyviolacein in sample group with blue light induction and group without blue light induction. Red column stands for yield of deoxyviolacein; Blue column stands for yield of violacein. Fig. B shows the HPLC result from sample without blue light induction. Fig. C shows the HPLC result from sample with from sample under blue light induction.The HPLC results show that the peak of deoxyviolacein appears at about 9 minutes after the injection. The peak of deoxyviolacein is identified according to previously published research. Results indicate that the amount of the deoxyviolacein from the sample without blue light induction is tremendously larger than that from light induced sample


Under the induction of blue light, production of deoxyviolacein was inhibited (to almost 0%) while most products were violacein, which is predicted above. Without blue light induction, however, the pathway leading to the production of deoxyviolacein was turned on and thus considerable amount of deoxyviolacein was produced. All results confirmed the feasibility of our light-sensing Membrane Rudder. Besides, it indicated that by replacing signal sensors and functioning enzymes, we could regulate direction of various branched reactions through diversified signals.

Decrease in Side Products

We also found membrane anchored enzymes could help to reduce the amount of side-product(deoxychromaviridans) by 8 fold compared to diffusing cytoplasmic enzymes. This is because rationally designed enzyme assembly facilitate and improve the metabolic flux of biosynthesis. Intermediate PVA is more preferably processed by adjacent membrane anchored VioD or VioC rather than converted into deoxychromaviridans. Actually this is one of the advantages of Membrane Scaffold.

Fig.5 :The HPLC peak area of deoxychromoviridans from sample with free VioABCDE(denoted as Free) and membrane anchored VioABCDE(denoted as Complex). Construction detail of membrane anchored VioABCDE is shown in Fig.2. Bacteria are incubated under light for 6 hours in Complex group.

Reference

1. Balibar, C. J. and C. T. Walsh (2006). "In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum." Biochemistry 45(51): 15444-57.

2. Hoshino, T. "Violacein and related tryptophan metabolites produced by Chromobacterium violaceum: biosynthetic mechanism and pathway for construction of violacein core." Appl Microbiol Biotechnol 91(6): 1463-75.

3. Shrode, L. B., Z. A. Lewis, et al. (2001). "vvd is required for light adaptation of conidiation-specific genes of Neurospora crassa, but not circadian conidiation." Fungal Genet Biol 32(3): 169-81.




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