Difference between revisions of "Part:BBa K5291033"

Revision as of 03:23, 2 October 2024

pAB1-alkB₂-Rd45-adhA

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Usage and Biology

AlkB2-Rd45-AdhA is designed to accelerate the degradation rate by improving the absorbing and hydroxylation of PE monomer. In order to express above the enzymes in our chassis，pAB1-AlkB2-Rd45-VHB was constructed.

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Fig.1 The map of the plasmid pAB1-AlkB2-Rd45-AdhA.

We transferred the constructed plasmid into Escherichia coli DH5αstrain and conducted colony PCR. After obtained the correct result we amplified and extracted the constructed plasmids. Then we transferred these plasmids into Pseudomonas aeruginosa PAO1 strain and E. coli BL21 strain and obtained correct colony PCR results, indicating that we successfully constructed strains containing the plasmid pAB1-AlkB2-Rd45-AdhA.

Fig.2 The P.aeryginosa PAO1 colony PCR results of AlkB2-Rd45-AdhA.

In order to verify whether AlkB2-Rd45-AdhA proteins was successfully expressed in the strains, SDS-PAGE was performed and the following results were obtained.

Fig.3 SDS-PAGE result of AlkB2-Rd45-AdhA.

Plasmid pAB1-AlkB2-Rd45-AdhA is expected to express a large fusion protein AlkB2-Rd45-AdhA with a molecular weight of 94.59kDa. In the figure, it can be seen that there were an extra bands in the experimental group than in the control group at a position slightly less than 100kDa, suggesting that AlkB2-Rd45-AdhA protein was successfully expressed.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 902
Illegal NgoMIV site found at 2433
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1476

@@ Line 7: / Line 7: @@
 ===Usage and Biology===
-AlkB2-Rd45-AdhA is designed to accelerate the degradation rate by improving the absorbing and hydroxylation of PE monomer. In order to express above the enzymes in our chassis， pAB1-AlkB2-Rd45-VHB was constructed.<br><br>
+AlkB2-Rd45-AdhA is designed to accelerate the degradation rate by improving the absorbing and hydroxylation of PE monomer. In order to express above the enzymes in our chassis，pAB1-AlkB2-Rd45-VHB was constructed.<br><br>
 <html><img width = "700" src="" /></html>(lack)
 <br><b>Fig.1 The map of the plasmid pAB1-AlkB2-Rd45-AdhA.</b><br><br>
-We transferred the constructed plasmid into <i>Escherichia coli</i> DH5αstrain and conducted colony PCR. After obtained the correct result we amplified and extracted the constructed plasmids. Then we transferred these plasmids into <i>Pseudomonas aeruginosa</i> PAO1 strain and <i>E. coli</i> BL21 strain and obtained correct colony PCR results, indicating that we successfully constructed strains containing the plasmid pAB1-AlkB2-Rd45-AdhA.<br><br>
+We transferred the constructed plasmid into <i>Escherichia coli</i> DH5αstrain and conducted colony PCR. After obtained the correct result we amplified and extracted the constructed plasmids. Then we transferred these plasmids into <i>Pseudomonas aeruginosa</i> PAO1 strain and <i>E. coli</i> BL21 strain and obtained correct colony PCR results, indicating that we successfully constructed strains containing the plasmid pAB1-AlkB2-Rd45-AdhA.<br>
 <html><img width = "350" src="https://static.igem.wiki/teams/5291/images/part-wyn/alkbcyp-colony-pcr.png" /></html>
 <br><b>Fig.2 The <i>P.aeryginosa</i> PAO1 colony PCR results of AlkB2-Rd45-AdhA.</b><br><br>
+In order to verify whether AlkB2-Rd45-AdhA proteins was successfully expressed in the strains, SDS-PAGE was performed and the following results were obtained.<br>
+<html><img width = "450" src="https://static.igem.wiki/teams/5291/images/part-wyn/sdspage-alkb-adh.png" /></html>
+<br><b>Fig.3 SDS-PAGE result of AlkB2-Rd45-AdhA.</b><br><br>
+Plasmid pAB1-AlkB2-Rd45-AdhA is expected to express a large fusion protein AlkB2-Rd45-AdhA with a molecular weight of 94.59kDa. In the figure, it can be seen that there were an extra bands in the experimental group than in the control group at a position slightly less than 100kDa, suggesting that AlkB2-Rd45-AdhA protein was successfully expressed.
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