Part:BBa_K4427002
LP，Our favourite part, which can output phenethyl alcohol
In this plasmid, we have added the EL222 photosensitive protein, which activates the expression of downstream genes when exposed to 465nm blue light, we have added the Adh1 and KdcA protease genes mentioned above. pSB1C3-stuffer-LP.dna is the complete plasmid system for our blue light system.
To ensure that the EL222 protein gene (BBa_K4427003), the Adh1 gene (BBa_K4427004) and the KdcA gene (BBa_K4427005) were inserted in the correct positions and the plasmids were correctly transformed, we performed plasmid extraction, double digestion (XbaI and SpeI), and agarose gel electrophoresis on DH5α bacteria solution after transformed with the pSB1C3-stuffer-Lp(BBa_K4427002). The results of the electrophoresis after plasmid extraction showed that our plasmid was successfully transformed into BL21, and the results of the XbaI and SpeI double digestion experiments showed that our target gene was inserted into the target site correctly.
The result of agarose gel electrophoresis after double digestion. Lane 1: pSB1C3-stuffer-Lp (lp) plasmid; Lane 2: DNA segments after digestion.
After determining that the plasmid was correctly constructed and successfully transformed, we verified the light-sensitive function of the pSB1C3-stuffer-Lp plasmid (BBa_K4427002) in a dark room. With reference to the literature, we performed periodic experiments on engineered bacteria BL21 in a dark room with blue light irradiation for 2 hours and duck treatment for 2 hours alternatively, for a total of 8 hours, and a control experiment was conducted in complete darkness. The experimental results showed that the engineered bacteria containing the pSB1C3-stuffer-Lp plasmid (BBa_K4427002) successfully expressed 2-PE, which validated the effectiveness of our blue light-controlled system.
We performed SDS-PAGE experiments on the two groups of bacterial solution, obtained the following figure (Fig. 16). The molecular weight of Adh1 is 37 kDa and KdcA is 62 kDa, which is comparable to the molecular weight of the Marker corresponding to the bands in the electropherogram, which can tentatively prove that the pSB1C3-stuffer-Lp plasmid (BBa_K4427002) successfully expresses the target protein we need.
The above experiments demonstrated that E. coli BL21 transfected with pSB1C3-stuffer-Lp plasmid (BBa_K4427002) could catalyze the synthesis of the substrate phenylpyruvate into 2-PE. We then performed the final product expression experiments[3], and compared the samples with the standard 2-PE. The following results were obtained, identifying our samples as 2-PE.
Gas chromatography of Standard 2-PE sample
Gas chromatography of the bacterial extract sample
2-PE cleavage mechanisms in electron bombardment ion sources:
Mass spectra of standard 2-PE samples
Mass spectra of extracted 2-PE samples in bacterial broth
The results of gas chromatography and mass spectrometry showed that the bacteriophage extract samples contained 2-PE, indicating that the overall blue light expression system was successful. We also used liquid chromatography to roughly determine our 2-PE yields as follows.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal suffix found in sequence at 4820
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 4821
Illegal PstI site found at 4835
Illegal NotI site found at 7
Illegal NotI site found at 4828 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1
Illegal BamHI site found at 1165
Illegal BamHI site found at 4743
Illegal XhoI site found at 1743
Illegal XhoI site found at 4752
Illegal XhoI site found at 5852
Illegal XhoI site found at 6744 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 4821 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 1
Illegal XbaI site found at 16
Illegal SpeI site found at 4821
Illegal PstI site found at 4835
Illegal NgoMIV site found at 1313
Illegal AgeI site found at 1000 - 1000COMPATIBLE WITH RFC[1000]
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