Plasmid

Part:BBa_K4427016

Designed by: Chanjuan Jin   Group: iGEM22_JLU_China   (2022-09-30)


Pet-22b-RLP 

The Red Light-Responding Switch

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In the red light-responding switch, we constructed pET-22b-RLP ( BBa_K4427016), which includes Ho1 (BBa_K4427010), the red light sensitive protein BphP1 (BBa_K4427000) and the deterrent protein PpsR2 (BBa_K4427001). It expresses Ho1, BphP1 and PpsR2 under the induction of IPTG. 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. To validate the above function of our red light initiation system pET-22b-RLP (BBa_K4427016), we performed the following validation

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:

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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

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

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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

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Fluorescence spectra of the experimental group with red light irradiation

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For our project, the red light repression function of the BphP1-PpsR2 system was not indispensable. Due to time constraints, we only performed experimental validation of the activated expression function and did not complete functional validation of the repressed expression function.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 5536
    Illegal EcoRI site found at 8352
    Illegal XbaI site found at 5163
    Illegal XbaI site found at 5996
    Illegal XbaI site found at 8116
    Illegal PstI site found at 1140
    Illegal PstI site found at 6520
    Illegal PstI site found at 6817
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 5536
    Illegal EcoRI site found at 8352
    Illegal PstI site found at 1140
    Illegal PstI site found at 6520
    Illegal PstI site found at 6817
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 5536
    Illegal EcoRI site found at 8352
    Illegal BglII site found at 5097
    Illegal BglII site found at 9494
    Illegal BamHI site found at 6176
    Illegal XhoI site found at 10568
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 5536
    Illegal EcoRI site found at 8352
    Illegal XbaI site found at 5163
    Illegal XbaI site found at 5996
    Illegal XbaI site found at 8116
    Illegal PstI site found at 1140
    Illegal PstI site found at 6520
    Illegal PstI site found at 6817
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 5536
    Illegal EcoRI site found at 8352
    Illegal XbaI site found at 5163
    Illegal XbaI site found at 5996
    Illegal XbaI site found at 8116
    Illegal PstI site found at 1140
    Illegal PstI site found at 6520
    Illegal PstI site found at 6817
    Illegal NgoMIV site found at 137
    Illegal NgoMIV site found at 3317
    Illegal NgoMIV site found at 3477
    Illegal NgoMIV site found at 5065
    Illegal NgoMIV site found at 6127
    Illegal NgoMIV site found at 6162
    Illegal NgoMIV site found at 7410
    Illegal AgeI site found at 9971
  • 1000
    COMPATIBLE WITH RFC[1000]


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