Part:BBa_K1779200
Integration of three genes, including mamW, RFP and laccase
Integration of three genes, including mamW, RFP and laccase. The mamW gene is isolated from Magnetotactic bacterium MSR-1. Its protein can be used as an anchor protein. RFP gene is extracted from iGEM standard biobrick BBa_E1010. And we used it as a reporter gene which expression can visualize the fusion protein in our experiment. The laccase gene which expressed oxidase enzyme is obtained from iGEM standard biobrick BBa_K863005. We used it into our experiment as the main substance on cathodic reaction in the biofuel cell.
1. We respectively amplified three genes by PCR.
Figure 1| The image of agarose gel electrophoresis.M: DNA marker, mamW and RFP and laccase were amplified by PCR using high fidelity DNA polymerase.
2. We respectively combined W+R+L by fusion PCR.
Figure 2| The image of agarose gel electrophoresis.M: DNA marker,W + R + L: The fusion PCR of mamW + RFP + laccase using high fidelity DNA polymerase.
3. We purified the PCR products and successfully inserted the genes into pACYCDuet-1, and named it as piGEM-WRL. Then we verified them using digestion and sequencing.
Figure 3|(1) The sample without restriction endonuclease. (2) The vector digested by PstI +XhoI.
4. We transferred piGEM-WRL into BL21 (DE3) and conducted the inducible expression. Then we got a series of samples which were cultivated at different time points. And we chose one to compare with empty vector of BL21 (DE3) as a control group.
Figure 4| The left is untransformed BL21 (DE3) and the right is transformed BL21(DE3) with piGEM-WRL.
5. Using Ultrasonic Cell Disruptor to crush the bacterium in ice-bath. Collecting the Supernatant and detect the activity of laccase by ABTS method.
Figure 5| Enzyme activities from different supernate using ABTS method (http://2015.igem.org/Team:CHINA_CD_UESTC/Protocol). Each supernate presents the different bacterium which are cultivated at different time points.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1005
Illegal AgeI site found at 1117 - 1000COMPATIBLE WITH RFC[1000]
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