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

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<div align="center"><img width="550px" src="http://2014.igem.org/wiki/images/f/fc/VUPV_Gb2.png" alt="solid_phase_extraction" title="Figure 2. Part categories of a basic GoldenBraid trancriptional unit. Promoter’s (PROM) prefix is GGAG and its suffix is AATG, which is the same as the coding region’s (CDS) prefix. The same happens with the CDS and the terminator (TER), which share the part identity overhang GCTT, the first one as its suffix and the second one as its prefix."></img></div><br/>
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<div align="center"><p style="text-align: center; font-size: 0.8em; width: 670px;"><b>Figure 2</b>. Part categories of a basic GoldenBraid trancriptional unit. Promoter’s (PROM) prefix is GGAG and its suffix is AATG, which is the same as the coding region’s (CDS) prefix. The same happens with the CDS and the terminator (TER), which share the part identity overhang GCTT, the first one as its suffix and the second one as its prefix.</p></div><br/>
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Revision as of 00:14, 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.



solid_phase_extraction

Figure 2. Part categories of a basic GoldenBraid trancriptional unit. Promoter’s (PROM) prefix is GGAG and its suffix is AATG, which is the same as the coding region’s (CDS) prefix. The same happens with the CDS and the terminator (TER), which share the part identity overhang GCTT, the first one as its suffix and the second one as its prefix.








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