Coding
Ppsr2

Part:BBa_K4427001

Designed by: Quan Luo   Group: iGEM22_JLU_China   (2022-09-20)


PpsR2


PpsR2, a repressor protein that is regulated by the expression of BphP1, i.e. when irradiated with 760 nm red light the conformation of Bphp1 is altered so that it inhibits the repressive effect of the repressor protein, i.e. the transcription of the promoter. 

 im-001-1.png

As shown in this figure, Ho1 catalyzes heme to BV, BV binds to BphP1 and acquires light-sensitive ability, and it can bind to PpsR2 under 760nm NIR light irradiation to inhibit the suppression of PpsR2, highly expresses downstream genes. While under 680nm red light irradiation, the suppression effect is quickly disabled and suppressed the expression of the downstream gene again. 

The result of agarose gel electrophoresis after double digestion.Lane 1: DNA segments after digestion; Lane 2: pET-22b-RLP (Redlight promotor) plasmid:

improve-part-001-2.png


After demonsrating that the plasmid was correctly constructed and successfully transformed, we verified the light-sensitive function of it in a dark room. With reference to the literature, we performed irradiation experiments experiments on engineered bacteria BL21 with far-red light irradiation for 4 hours and a control experiment with complete light-proofing. After the irradiation, the following observations were made on the bacterial solution.

We used a UV lamp to illuminate the two groups of bacterial fluids and the results showed that clear fluorescence appeared in the experimental group, while no fluorescence appeared in the control group. 

Comparative graph of fluorescent protein under UV irradiation, control A1 did not fluoresce and red light irradiated group A2 showed significant fluorescence

improve-part-001-3.png 

Comparison graph under the naked eye, control group A1 is colourless, red light irradiated group A2 appears light green

improve-part-001-4.png


To determine whether the protein expressed by the engineered bacteria was the green fluorescent protein (sfGFP) that we expected, we performed fluorescence spectroscopy on the bacterial solution. The results showed the protein had an excitation wavelength of 488 nm and an emission wavelength of 510 nm, which is consistent with the standard sfGFP. It determined that our engineered bacteria synthesized sfGFP successfully. However, the results of the fluorescence spectra of the bacterial broth in the light-avoidance group also showed that sfGFP was synthesized and that leakage of gene expression may have occurred. 

Fluorescence spectrum of the control solute without red light irradiation, protected from light

improve-001-5.png 

Fluorescence spectra of the experimental group with red light irradiation

improve-001-66.png 


In the previous iGEM teams, they used the former version of the PpsR2, and not only one team used this protein.
However, none of them had successful results.
Then, we change QPAS1 to PpsR2, and we got promising experiment results, which can be beneficial for future iGEM teams.
The former version is BBa_K4060506
And for more information, you can find it in our wiki.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 208
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 208
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 208
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 208
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 208
    Illegal NgoMIV site found at 91
    Illegal NgoMIV site found at 406
    Illegal NgoMIV site found at 1241
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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