Team:USP-UNESP-Brazil/Project

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(Overall project)
(Plug&Play Plasmid)
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=== Plug&Play Plasmid ===
=== Plug&Play Plasmid ===
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The Plug&Play machine propose aims to construct a tool for a faster protein
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Aware of the necessity of techniques to produce biological standardize parts in a high throughput manner we developed a project that aims to build a machine, called Plug&Play, that express any protein using the Cre-Recombinase system. For this purpose, we designed a plasmid that has a mutated recombination site for the Cre-recombinase, in which the desired ORF (open reading frame) is going to be inserted, upstream the insertion site is located a promoter ready to transcript the gene into the desire protein. The plasmid has also, a resistance gene to ampicillin that maintain it inside the cell as long as the antibiotic is applied in the culture medium. This is a high throughput system for expressing proteins that allows putative (or new build) genes prospection, which is mean to be an open source tool.
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expression, what would allow to accelerate the choice of genes of interest. The main idea is to
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create an open source set of genes for various expression cases, homologous to the different
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expression vectors available on the market. As proof of concept, is proposed the genesis of a
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plasmid that could allow the expression of any protein inside E.coli in two passages: PCR
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(Polimerase Chain Reaction) and transformation.
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The main concept which the project relies on is the Cre recombinase protein action.
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This enzyme can accelerate the recombination between specific sites known as loxP, whose
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sequence have two ligation sites allowing recombination inside the chromosomes. By this
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action the Plug&Play system is composed of a pair of primers to amplify the ORF sequence
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of a given protein. Each set of primers have the sequence capable of recognize the ORF flanked
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by one of the assembling sites of loxP site. This assembling sequences will have two main
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functions: circularize the PCR product on the moment it enters on the bacteria and the
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recombine with a new plasmid that will be inside the bacteria. This plasmid is put in the
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bacteria, allowing it to accept any PCR amplified ORF flaked by loxP sites, using the
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recombination mechanism (Cre recombinase) to insert the sequence on the right place,
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allowing to the gene to become susceptible to the transcription machinery
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For more information about this project,[[Team:USP-UNESP-Brazil/Project2| click here! ]]
For more information about this project,[[Team:USP-UNESP-Brazil/Project2| click here! ]]

Revision as of 15:56, 15 September 2012

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Contents

Synthetic Biology

Synthetic Biology is an emerging field that aims to modify organisms to perform new tasks by either constructing new biological parts, devices and systems or by re-designing an already existing system turning it into a useful purpose for humankind.

The difference to classical molecular biology is the use of engineering concepts, as complexity abstraction and standardization of parts, into the design of molecular circuits. This approach allows to produce more reliable systems build from bottom to top (from genetic circuits to complex metabolic pathways), allowing to create more complex systems that the already known in classical molecular biology. This field requires easy access to standardized biological parts and devices, well-known cells where DNA programs can be assembled and powered, as well as, computational tools to analyze the created systems.


Overall project

Since we are a big team and it's our first participation on iGEM, we have two projects going on at the same time. The first one is "Associative Memory Network Using Bacteria" and "Plug&Play Plasmid"

Associative Memory Network Using Bacteria

The memory storage in biological systems has a critical role in biotechnology development. A systemic way of storing a specific memory that can be recovered and used at any moment is studied in several experiments and mathematical models involving neural networks. One of these models, known as “Hopfield Network”, considers the memory storage as a neurons association that shares a characteristic pattern of “communication intensity” – the “measure unity” of a neuron network . This model is notorious for allowing systems the recognizing of patterns.

In this project we propose the genesis of aa communication network using E.coli populations, in order to create a system with associative memory ,like a Hopfield Model. Genetically different populations will be generated and isolated from each other, keeping contact by means of Quorum Sensing Substances (QSS). These QSS will be responsible by inhibition or excitation of pre-determined populations, measuring the amount of excitation by means of GFP fluorescent level. The final objective is to achieve a specific complete pattern of excited and inhibited populations by means of the interactions between bacteria populations, based on an given incomplete pattern. The network will behave in way of recognizing this pattern and choose between two systemic memories inserted on the communication network.

For more information about this project, click here!

Plug&Play Plasmid

Aware of the necessity of techniques to produce biological standardize parts in a high throughput manner we developed a project that aims to build a machine, called Plug&Play, that express any protein using the Cre-Recombinase system. For this purpose, we designed a plasmid that has a mutated recombination site for the Cre-recombinase, in which the desired ORF (open reading frame) is going to be inserted, upstream the insertion site is located a promoter ready to transcript the gene into the desire protein. The plasmid has also, a resistance gene to ampicillin that maintain it inside the cell as long as the antibiotic is applied in the culture medium. This is a high throughput system for expressing proteins that allows putative (or new build) genes prospection, which is mean to be an open source tool.

For more information about this project, click here!

Project Details

Part 2

The Experiments

Part 3

Results