Team:Brown/Project

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Latest revision as of 21:07, 27 October 2010

Light-Pattern Controlled Circuit

Abstract

Biological manufacturing of complex compounds often requires the synthesis of many intermediate products. Production of these intermediates is currently triggered by inefficient methods, such as chemical inputs (tetracycline, estrogen-analogs, arabinose, etc) or drastic changes to the cellular environment (pH, oxygen levels, temperature, etc). On an industrial scale, this chemical induction requires large quantities of reagents and extensive purification, while environmental induction requires conditions that can adversely affect cell vitality and yield. To this end, we are engineering an E. coli genetic circuit that can pass through four stable states of protein production triggered solely by ON/OFF patterns of light. With this production method, we can link multiple synthesis steps to a single, clean and rapidly scalable input.

E. Cargo

Abstract

We designed a modular Tat-Linker Biobrick that will allow for easy fusion of the Tat-PTD to any other biobricked proteins. Specifically, we aimed to fuse this Tat-Linker in RFC25 format to two bacterial transcription factors, LacI and AraC, with the intent of using the two Tat-TFs as a tool to induce transient gene expression in E. coli without the need to follow through with more time-consuming cell transformation protocols. To test these Tat-TFs, we designed corresponding reporter constructs. We saw a potential application in the portion of the Light-Pattern Controlled Circuit of our project, as well as in any other future genetic circuits that require part-by-part testing.

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-<!--- The Mission, Experiments --->
+{{:Team:Brown/templates/header}}
-{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
+<br>
-!align="center"|[[Team:Brown|Home]]
-!align="center"|[[Team:Brown/Team|Team]]
-!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Brown Official Team Profile]
-!align="center"|[[Team:Brown/Project|Project]]
-!align="center"|[[Team:Brown/Parts|Parts Submitted to the Registry]]
-!align="center"|[[Team:Brown/Modeling|Modeling]]
-!align="center"|[[Team:Brown/Notebook|Notebook]]
-!align="center"|[[Team:Brown/Safety|Safety]]
-|}
-== '''Project 1 - TAT-PTD''' ==
-=== Project Description ===
+== '''Light-Pattern Controlled Circuit''' ==
-We have created a recombinant protein which fuses a Tat-protein transducing domain (Tat-ptd) with a synthetic single chain variable fragment (scFv) domain. The goals of this project are: 1) be able to stably express the recombinant protein in E. Coli; 2) create a protein that will retain conformation and function in cytosolic conditions; 3) the intrabody should be able to bind with high specificity to the desired target protein; 4) the intrabody can translocate across mammalian cell and tissue barriers; and 5) include a method for easy fluorescence reporting.
+'''Abstract'''
-We also are in the process of creating a biobrick that will allow for easy fusion of the Tat-PTD to another biobricked protein. This will allow researchers and teams to administer TAT-tagged transcription factors for transient activation/repression of target genes.
+Biological manufacturing of complex compounds often requires the synthesis of many intermediate products. Production of these intermediates is currently triggered by inefficient methods, such as chemical inputs (tetracycline, estrogen-analogs, arabinose, etc) or drastic changes to the cellular environment (pH, oxygen levels, temperature, etc). On an industrial scale, this chemical induction requires large quantities of reagents and extensive purification, while environmental induction requires conditions that can adversely affect cell vitality and yield. To this end, '''we are engineering an E. coli genetic circuit that can pass through four stable states of protein production triggered solely by ON/OFF patterns of light.''' With this production method, '''we can link multiple synthesis steps to a single, clean and rapidly scalable input.'''
+<html>
+<center>
+<a href="https://2010.igem.org/Team:Brown/Project/Light_pattern/Overview"><img src="https://static.igem.org/mediawiki/2010/thumb/c/c2/LRCpeektacropped.jpg/780px-LRCpeektacropped.jpg" width="500px"></a>
+</center>
+</html>
-=== Progress ===
+== E. Cargo ==
-== '''Project 2 - Quad-state light-activation''' ==
+'''Abstract'''
-=== Project Description ===
+We designed a modular Tat-Linker Biobrick that will allow for easy fusion of the Tat-PTD to any other biobricked proteins. Specifically, we aimed to fuse this Tat-Linker in RFC25 format to two bacterial transcription factors, LacI and AraC, with the intent of using the two Tat-TFs as a tool to induce transient gene expression in <i>E. coli</i> without the need to follow through with more time-consuming cell transformation protocols. To test these Tat-TFs, we designed corresponding reporter constructs. We saw a potential application in the portion of the Light-Pattern Controlled Circuit of our project, as well as in any other future genetic circuits that require part-by-part testing.
+<html>
-The aim of this project is to create a cell that can go through a sequence of four distinct states in response to a timed pattern of light-on, light-off.
+<center>
+<a href="https://2010.igem.org/Team:Brown/Project/Ecargo/Overview"><img src="https://static.igem.org/mediawiki/2010/thumb/8/8d/ECargo.png/400px-ECargo.png" width="500px"></a>
-This project makes use of parts from EPF Lusanne '09 (LOVTAP light activated promoter), PKU '07/'09 (bistable switch, AND gate), and Missouri Western State University '08 (Hybrid promoter). Our goal is to show that it is possible and easy to create entirely new cell logic systems from parts that already exist in the registry.
+</center>
+</html>
-See the model for this system here:[[Team:Brown/Modeling]]
-=== Progress ===
-== '''Project 3 - Miracle Yogurt''' ==
-=== Background ===
-In the early eighteenth century, French cartographer and navigator Chevalier des Marchais travelled all along the west coast of Africa. His maps and manuscripts were published after his death by Père J. B. Labat in Amsterdam in 1730-31. One of these, from 1725, documented a peculiar food culture among local tribes, who consumed tiny red berries before most meals.
-These berries are known as Synsepalum dulcificum, Richadella dulcifica, or colloquially ‘miracle fruit’. They have a peculiar quality: for 30 minutes to two hours after consumption, sour foods are perceived as sweet. This fascinating property was not rediscovered until the mid-19th century, when Prof. Kenzo Kurihara, a Japanese scientist, published an article in Science about the isolation of the active compound, which he coined miraculin.
-Soon afterward, Robert Harvey and Don Emery became co-founders of a miracle berry start-up called Miralin. In 1974, the FDA cut support for the company and halted the approval of miracle berries as a harmless food additive only weeks after the Miralin offices were raided by an unknown party. Although the identity of the thieves was never determined, claims have been made that the raid and subsequent FDA disapproval were supported by high-ups in the sugar and sweetener industry.
-Today, miracle berries are making an underground comeback thanks to a few small startups, with slogans such as mBerry’s “Make life sweeter.” In the last few years, their popularity has grown exponentially, and it’s all thanks to a single protein named miraculin.
-Miraculin is a small glycoprotein, 191 amino acids long, that was first sequenced in 1989. Almost 14% of the protein’s weight is made up of various sugars, but these are not necessary for the taste-modifying effects. In miracle berries, miraculin occurs as a tetramer, more specifically a combination of two homodimers. Within these dimers, two miraculin monomers are bound together by a disulfide bridge. Miraculin is readily soluble in aqueous solution and is heat stable up to 100°C. It is active at pH 3-12, and can remain stable and active at pH 4 for 6 months in a refrigerated environment.
-Unlike similar taste-modifying proteins, miraculin does not intrinsically possess a sweet taste, however the maximum sweetness response induced by miraculin has been shown equivalent to 17% sucrose solution (very sweet).  The mechanism of miraculin’s taste-modifying properties has not yet been conclusively determined. Mutation experiments suggest that two histidine residues (His29 and His59) are mainly responsible for these properties. Both histidine residues are involved in dimerization. It is hypothesized that one site acts to attach miraculin to the cell membranes of the sweet tastebuds. The other induces a conformational change in response to protonation (acids, the major components of sour taste, are proton donors) that brings miraculin in contact with an active site of a receptor on the surface of the tastebud cell.
-Together, these cause miraculin to bind transiently to the tastebud and induce activation of the sweetness response when in the presence of sour acids. The ability of the fruit to turn non-sweet foods into sweet foods without a caloric penalty is highly valuable, and alone provides a reason to mass-produce miraculin-containing miracle fruit. Synsepalum dulcificum, however, is not a practical plant to mass-produce. Its seeds have a 24% sprouting success rate, and the young plants require high humidity and partial shade. They do not bear fruit for the first 2-3 years, and only crop out twice a year.  What’s more, it takes 2-3 berries worth of pulp to yield one ‘dose’. These limitations make miraculin an excellent candidate for production in transgenic organisms.
-If miraculin, tagged with a secretion signal, can be successfully inserted into L. lactis, it will result in an extraordinarily cheap and easily distributable mechanism for the mass-production of miraculin-containing yogurt, buttermilk, or cheese. To make yogurt, a small amount of active yogurt or dehydrated lactobacillus is added to milk and incubated at 38C for seven hours. Dehydrated bacteria can be easily shipped by mail, and a small amount of previously-made yogurt can be used as a starter culture for further production.
-This yogurt would prove valuable for a number of populations: diabetics who cannot consume overly sugary foods, dieters who want to lose weight but cannot stick to their bland diets, cancer patients whose chemotherapy leaves a bitter, metallic taste (a pilot study has indicated that miracle berries counteract this effect), children who cannot stand the taste of bitter/sour foods, and foodies who seek a novel perceptual experience. By increasing consumption of probiotic cultures and decreasing consumption of high-calorie sugars and carcinogenic artificial sweeteners such as aspartame, the mass production and distribution of a miraculin-containing yogurt could provide a healthier lifestyle.
-=== Project Description ===
-We plan to insert the miraculin gene, along with a secretion tag, into Lactococcus lactis. This will be done via a hybrid E.coli/Lactobacillus shuttle vector pPTPi.