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Team:NCTU Formosa/introduction
From 2011.igem.org
Introduction
Our program is to design “Pathway Commander”, just like its name, which is able to direct a pathway’s going. The goal is to control a pathway to stop at any intermediate step. In traditional pathway design, one circuit will express itself all to the end, unable to stop at any step involved. However, with our “RNA Thermometer”, which is temperature-controlled, the pathway can be easily controlled at specific temperature. The circuit we design can stop at any intermediate state we want and accumulate intermediate products, and then continue to express if the condition is changed. Even branched circuit can also be created.
Our team put the new idea in use to improve the production of butanol which is catalyzed by alpha-ketoisovalerate decarboxylase encoded by kivd gene. We use low-temperature-induced pathway because our product will easily vaporize in high temperature, and the specific enzyme involved has better activity at 30℃. We know that butanol is fatal to E.coli if the amount is above 2%. Now, we can exploit our “Pathway Commander” idea to the fullest in this situation. First, intermediate products accumulate until enough quantity to be bio-fuel and then we lower the temperature to induce the enzyme which catalyzes precursors into butanol. (Figure 1.)Of course, the E.coli dies due to butanol of high concentration, and there won’t be environmental issue and get enough butanol to produce bio-fuel!
Figure 1: The reporter circuit: This isobutanol metabolic pathway applies the circuits BBa_K539742 and BBa_K539691 made by our team. When the temperature is at 37℃, alss, ilvC and ilvD are turned on, so the precursors go through a catalytic cascade and finally a large amount of 2-Ketoisovalerate is accumulated. 2-Ketoisovalerate is not poisonous to E.coli . Later, lower the temperature to 30℃ and kivd is turned on and Kivd catalyzes accumulative 2-Ketoisovalerate into large amount of butanol . E.coli cannot bear this amount of butanol and die. In the end, this temperature-induced method improves the quantity of butanol.
We also apply “Pathway Commander” to other pigment metabolic pathway in order to test the idea of temperature-induced method. We design two examples to demonstrate our idea, which are Carotenoid pathway and Volacein pathway.
In Carotenoid pathway, we can stop the pathway at three states. In every state, it can turn to the next state after accumulate enough intermediates. For instance, when the temperature is below 37℃, E.coli synthesizes enzyme CrtE,CrtB and CrtI, which catalyze the colorless farnesyl pyrophosphate into pigment-Lycopen, and the red color is displayed. Later, when the temperature rises to 37℃, E.coli expresses crtY which catalyzes accumulative Lycopen into β-carotene, which shows obvious orange color. Finally, we raise the temperature up to 42℃ and E.coli expresses crtZ which catalyzes β-carotene into Zeaxanthin, and the yellow color is displayed consequently.(Figure 2.)
Figure 2: Carotenoid pathway applies the circuits BBa_K539151 and BBa_K539281. At 30℃, enzyme cassette crtEBI catalyze the colorless farnesyl diphosphate into pigment-Lycopene, and the red color is displayed. Later, when the temperature rises to 37℃, E.coli synthesizes CrtY which catalyzes accumulative Lycopene into β-carotene, which is orange. Finally, we raise the temperature up to 42℃ and E.coli synthesizes CrtZ which catalyzes β-carotene into Zeaxanthin, and the yellow color is shown.
In Violacein pathway, we bear the same idea in mind controlling the pathway with branches. Intermediate PVA can turn into Deoxychromoviridans or Protoviolaceinic acid regulated by different temperatures. The Protoviolaceinic acid will turn into violacein eventually and then two branches are created.(Figure 3.)
Figure 3: Violacein pathway . At 30℃, the colorless product called Protodeoxyviolaceinic acid(PVA) shows up. Then, If we raise the temperature up to 42℃ directly, the PVA would be catalyzed to Deoxychromoviridans by VioC (BBa_K539513). At the same time, our E.coli will turn into dark purple. On the other hand, if we raise the temperature up to 37℃ first, we would obtain dark green product called Protoviolaceinic acid catalyzed by VioD ( BBa_K539413). Then we cultivate the E.coli in 42℃ after placing in 37℃ for a while , the resulting product is violet Violacein catalyzed by VioC .
