Part:BBa_K4427009
pSB1C3-stuffer-pro
Verification of successful transformation of EL222 plasmid
As shown in the plasmid map, we have attached the red fluorescent protein mRFP1 to the EL222 protein, and we intend to confirm the successful transformation of the EL222 plasmid by co-transforming the plasmid with the control plasmid into E.coli and testing whether the plasmid can emit red fluorescence under fully shaded transformation conditions and when illuminated with 365nm UV light. The successful transformation of the EL222 plasmid was confirmed.
We performed functional validation of the pro plasmid in a dark room. With reference to the literature we performed cycles of blue light irradiation for 2 hours and dark hours under light-proof conditions for the engineered bacteria for a total of 8 hours in two rounds and a control experiment with complete light-proofing.
At the end of the two experimental cycles, the fluorescence was checked using UV light irradiation. The results of the experiment showed that the experimental group showed significant fluorescence, while the control group showed no fluorescence. (The graph on the left shows a comparison of the fluorescent proteins under UV irradiation, with the blue light irradiated group (left) showing significant fluorescence and the control group (right) showing no fluorescence. (The right graph shows the comparison under the naked eye, the blue light irradiated group (left) appears light pink, the control group (right) is colourless)
To determine whether the protein expressed by the engineered bacteria was the mRFP1 fluorescent protein that we expected, we performed fluorescence spectroscopy on the above bacterial broth that was determined to appear fluorescent by UV irradiation. The results of the experimental data showed that the experimentally obtained fluorescent protein had an absorption wavelength of 612 nm and an emission spectrum of 589 nm, which is consistent with the data related to standard mRFP1.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal suffix found in sequence at 2795
Illegal EcoRI site found at 1
Illegal XbaI site found at 16 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1
Illegal SpeI site found at 2796
Illegal PstI site found at 2810
Illegal NotI site found at 7
Illegal NotI site found at 2803 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
Illegal BglII site found at 1939
Illegal BamHI site found at 1165
Illegal BamHI site found at 2717
Illegal XhoI site found at 1743
Illegal XhoI site found at 2726
Illegal XhoI site found at 3827
Illegal XhoI site found at 4719 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 2796 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1
Illegal XbaI site found at 16
Illegal SpeI site found at 2796
Illegal PstI site found at 2810
Illegal NgoMIV site found at 1313
Illegal AgeI site found at 1000
Illegal AgeI site found at 2593
Illegal AgeI site found at 2705 - 1000COMPATIBLE WITH RFC[1000]
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