Difference between revisions of "Part:BBa K1789018"

 
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<partinfo>BBa_K1789018 short</partinfo>
 
<partinfo>BBa_K1789018 short</partinfo>
  
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This is a device which contains the sequence of the recombination of TALE1 and GFP1, the recombination of TALE2 and GFP2 and scaffold 1. Through this device, we can test and verify that our scaffold can strengthen the function of GFP.
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This is a device which contains the sequence of the recombination of TALE1 and GFP1, the recombination of TALE3 and GFP2 and scaffold 1. Through this device, we can test and verify that our system is effective and correct.
  
 
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===Usage and Biology===
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==Usage and Biology==
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Using the DNA-binding characteristic of the TAL effector, we can generate a new method to increase the production of heterogenous multi-enzymatic reactions in Prokaryotic cells by rationally designed TALE proteins fused with specific enzymes and their corresponding DNA sequences (as known as Binding Motifs [BMs]). Here we used Escherichia coli as our chassis. A plasmid backbone was used as the scaffold for those BMs. The same plasmid was used to encode the fusion protein. Thus, enzymes fused with TALE proteins could be gathered around the DNA scaffolds, enrich the local enzyme concentration, and promote the rate of reaction.
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This is the first experimental group of our project. Our theory is feasible if the functional parameter of this group is stronger than the group of negative control.
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Split GFP is a technique that has been widely used in the research of protein-protein interaction. In our project, we demonstrated a prototype by fusing the Amino (or Carboxyl) Half of GFP with TALE1 (or TALE2/3).
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By integrating the coding sequences of the TALE-fused proteins and the scaffold, three different plasmids can be constructed and this is the first one.
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[[File:GFP SCAF1.jpg]]
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Fig. 1 Split GFP fused with TALE1/TALE3 on SCAF1
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This prototype is designed to test if our system can achieve our goal of compartmentation by examining if the green florescent intensity raised observably.
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Fluoroskan Ascent FL by Thermo can be used to detect the fluorescence intensity.
  
 
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===Functional Parameters===
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<partinfo>BBa_K1789018 parameters</partinfo>
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==Experimental Validation==
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===Sequencing===
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This part is sequenced as correct after construction.
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===Split GFP Assay===
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===References===
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1. J. Boch, U. Bonas, Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annual review of phytopathology 48, 419 (2010).
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2. J. Boch et al., Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509 (Dec 11, 2009).
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3. M. J. Moscou, A. J. Bogdanove, A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (Dec 11, 2009).

Revision as of 09:57, 18 September 2015

GFP_S1

This is a device which contains the sequence of the recombination of TALE1 and GFP1, the recombination of TALE3 and GFP2 and scaffold 1. Through this device, we can test and verify that our system is effective and correct.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2311
    Illegal BamHI site found at 4908
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 5323
    Illegal AgeI site found at 5353
    Illegal AgeI site found at 5383
    Illegal AgeI site found at 5413
    Illegal AgeI site found at 5443
    Illegal AgeI site found at 5473
    Illegal AgeI site found at 5503
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1304
    Illegal BsaI.rc site found at 3390
    Illegal BsaI.rc site found at 3798
    Illegal BsaI.rc site found at 4104
    Illegal BsaI.rc site found at 5094