Coding

Part:BBa_K2510100

Designed by: Aya Gomaa   Group: iGEM17_Paris_Bettencourt   (2017-10-27)


pdDronpa wt

Dronpa is a reversible photoswitchable fluorescent protein that is switched on by default “fluorescent” and is switched off when illuminated by cyan light (~500nm). Dronpa Fluorescence is recovered by shining violet light (~400nm). And has been used in a design that facilitates the optical control of protein activities This part contains a device of two copies of Dronpa Fluorescent Protein that are codon optimized for E Coli with two BsaI cutting site in between to allow the insertion of various proteins to be tested

Mechanism of action:

The enzyme of interest is placed between 2 copies of Dronpa fluorescent protein via a linker. By switching on Dronpa (violet light) the protein of interest is caged as the two copies would dimerize. And by switching off Dronpa (cyan light), the two copies would dissociate which will result in activation of the protein of interest.


in our project this part has been tested with repressors and with beta gal enzyme


<img src="96_conditions.png"> 

                            Figure 1 :  the experiment conducted with the repressors caged with Dronpa
                                        
                                   </span>

                                     <img src="Repression_strength_of_closed_state.png ">

                                      Figure 2 :  An over view of the results of the cell-free experiment. Each 
                                       promoter was tested with its cognate repressors. Our results show that in 90% of our 
                                       constructs caging the repressors with Dronpa has increased the repression strength. 
                                       We have also obtained a wide linear range of repression strength-indicated by the red 
                                       slope-.
                                   </span>
                                   <img src="TetR_dronpa.png">
                                   <img src="P22c2_dronpa.png">
                                    <img src="HKCI_caged_dronpa.png"> 
                                   
                                           Figure 3: detailed Results of the cell-free experiment. Each promoter was 
                                           tested with its cognate repressors. Top: Testing with TetR caged with 
                                           either wt-Dronpa (BBa_K2510108) or a mutated version(BBa_K2510109)Middle: 
                                           Testing with P22c2 caged with either wt-Dronpa (BBa_K2510112) or a mutated 
                                            version(BBa_K2510113).Bottom: Testing with HKcI caged with either wt-
                                           Dronpa (BBa_K2510110) or a mutated version(BBa_K2510111)           

                                  <img src="Wtvs_mut_repressors.png" >
                                   Figure 4: the wtDronpa has a better control of HKC1 and C2P22 repressors while the mutDronpa has a better control of TetR repressors </span>


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 755
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 758
    Illegal BsaI.rc site found at 752


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