Team:Valencia UPV/Project/modules/methodology/gb

From 2014.igem.org

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<p><h3 class="hook" align="left"><a>Project</a> > <a href="http://2014.igem.org/Team:Valencia_UPV/Project/modules">Modules</a> > <a href="http://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology">Methodology</a> > <a href="http://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology#cloning">Cloning</a> > <a>GolgenBraid</a></h3></p><br/><br/>
<p><h3 class="hook" align="left"><a>Project</a> > <a href="http://2014.igem.org/Team:Valencia_UPV/Project/modules">Modules</a> > <a href="http://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology">Methodology</a> > <a href="http://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology#cloning">Cloning</a> > <a>GolgenBraid</a></h3></p><br/><br/>
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<div align="center"><span class="coda"><roja>T</roja>he <roja>G</roja>olden<roja>B</roja>raid cloning strategy</span> </div><br/>
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<div align="center"><span class="coda"><roja>T</roja>he <roja>G</roja>olden<roja>B</roja>raid <roja>c</roja>loning <roja>s</roja>trategy</span> </div><br/>
<p>Our Sexy Plant is a challenging project for many reasons; a very important one is that we use plants as chassis for engineering. Plants have eukaryotic gene structure, make use of plant-specific regulatory regions and require special T-vectors for transformation, among other special features. Consequently, DNA repositories and DNA assembly standards need certain adaptations to facilitate engineering using plant chassis. Without letting aside BioBricks, we decided to use the GoldenBraid system (GB) to build several of the intermediate genetic constructs employed in this project. GB is a DNA assembly system specially conceived to facilitate genetic engineering in Plant Synthetic Biology projects (visit <a href="http://gbcloning.org" class="normal-link-page" target="_blank">gbcloning.org</a> for more information). </p>
<p>Our Sexy Plant is a challenging project for many reasons; a very important one is that we use plants as chassis for engineering. Plants have eukaryotic gene structure, make use of plant-specific regulatory regions and require special T-vectors for transformation, among other special features. Consequently, DNA repositories and DNA assembly standards need certain adaptations to facilitate engineering using plant chassis. Without letting aside BioBricks, we decided to use the GoldenBraid system (GB) to build several of the intermediate genetic constructs employed in this project. GB is a DNA assembly system specially conceived to facilitate genetic engineering in Plant Synthetic Biology projects (visit <a href="http://gbcloning.org" class="normal-link-page" target="_blank">gbcloning.org</a> for more information). </p>
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<p>Type IIS restriction enzymes, unlike type II enzymes; cleave DNA at a defined distance from their recognition sites, not requiring any specific sequence in the cleavage site. Since there are no sequence requirements in the cleavage sites, these can be defined by the user and adapted to serve as standard fusion sites to DNA parts. The enzymes used in GoldenBraid are BsaI and BsmBI, which cut out from their binding sites generating 4 base overhangs.</p>   
<p>Type IIS restriction enzymes, unlike type II enzymes; cleave DNA at a defined distance from their recognition sites, not requiring any specific sequence in the cleavage site. Since there are no sequence requirements in the cleavage sites, these can be defined by the user and adapted to serve as standard fusion sites to DNA parts. The enzymes used in GoldenBraid are BsaI and BsmBI, which cut out from their binding sites generating 4 base overhangs.</p>   
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        <h3>Measurements</h3><br/>
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<p>Such a complex project as the Sexy Plant, requires many different measurement techniques. </p>
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<div align="center"><img width="550px" src="http://2014.igem.org/wiki/images/9/91/VUPV_Gb1.png" alt="solid_phase_extraction" title="Figure 1. Comparison between type II and type IIS restriction enzymes"></img></div><br/>
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<div align="center"><p style="text-align: center; font-size: 0.8em; width: 670px;"><b>Figure 1</b>. Comparison between type II and type IIS restriction enzymes</p></div><br/>
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<p>In order to analyse the pheromone production in the plant, we collected transformed Nicotiana benthamiana leaf samples and performed a <a href="http://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/sample_preparation" class="normal-link-page">Headspace SPME</a>, a technique that traps the volatile organic compounds produced in the sample. Then, the volatiles were analysed and identified by <a href="http://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/sample_analysis" class="normal-link-page">Gas Chromatography-Mass Spectrometry.</a></p>
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<h3> GoldenBraid step by step</h3> <br/>
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<h4> 1. GB Domestication </h4>
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The first step in the GB cloning strategy is the adaptation of the DNA sequence to the GB standard. This process is called domestication and implies (1) the removal of internal restriction sites for the enzymes used in GB (BsaI, BsmBI and BtgZI) and (2) the addition of appropriate 4-nt flanking overhangs to convert the DNA sequence into a standard part (Gbpart). Gbparts are the minimal standard building blocks and they are classified in different categories according to their specific function. </p>
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<p>Willing to test if the plants efficiently released the pheromone, we also performed a <a href="http://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/dynamic_headspace" class="normal-link-page"> Dynamic Headspace sampling technique.</a></p>
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<p>We also wanted to study moth’s response to pheromones produced by our genetically engineered plants. Therefore we performed an <a href="http://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/EAG" class="normal-link-page">Electroantennography</a> to test the antennae detection and signal transmission upon stimulation with our plant samples. In addition, we performed a <a href="http://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/windtunnel" class="normal-link-page">Wind tunnel assay</a> to observe male moths behaviour under stimulation with our pheromones.</p>
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<p>Finally, to test the induction of gene expression triggered by our cupper-activated switch, we performed a <a href="http://2014.igem.org/Team:Valencia_UPV/Project/modules/methodology/luciferase" class="normal-link-page">Luciferase expression assay</a>. </p>
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Revision as of 00:11, 18 October 2014


Project > Modules > Methodology > Cloning > GolgenBraid



The GoldenBraid cloning strategy

Our Sexy Plant is a challenging project for many reasons; a very important one is that we use plants as chassis for engineering. Plants have eukaryotic gene structure, make use of plant-specific regulatory regions and require special T-vectors for transformation, among other special features. Consequently, DNA repositories and DNA assembly standards need certain adaptations to facilitate engineering using plant chassis. Without letting aside BioBricks, we decided to use the GoldenBraid system (GB) to build several of the intermediate genetic constructs employed in this project. GB is a DNA assembly system specially conceived to facilitate genetic engineering in Plant Synthetic Biology projects (visit gbcloning.org for more information).


As BioBricks, GB is a modular cloning strategy that allows the fabrication of new devices by the combination of prefabricated standard modules. A difference between both strategies is that BioBricks is based on type II enzymes and GB relies on the use of type IIS restriction enzymes.


Type IIS restriction enzymes, unlike type II enzymes; cleave DNA at a defined distance from their recognition sites, not requiring any specific sequence in the cleavage site. Since there are no sequence requirements in the cleavage sites, these can be defined by the user and adapted to serve as standard fusion sites to DNA parts. The enzymes used in GoldenBraid are BsaI and BsmBI, which cut out from their binding sites generating 4 base overhangs.



solid_phase_extraction

Figure 1. Comparison between type II and type IIS restriction enzymes


GoldenBraid step by step


1. GB Domestication

The first step in the GB cloning strategy is the adaptation of the DNA sequence to the GB standard. This process is called domestication and implies (1) the removal of internal restriction sites for the enzymes used in GB (BsaI, BsmBI and BtgZI) and (2) the addition of appropriate 4-nt flanking overhangs to convert the DNA sequence into a standard part (Gbpart). Gbparts are the minimal standard building blocks and they are classified in different categories according to their specific function.









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