Part:BBa_K1779204
Integration of twogenes, including RFP and laccase
Integration of two genes, including RFP and laccase. 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 the 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 two genes by PCR.
Figure 1| We amplified ‘‘RFP’’ and Laccase by PCR using high fidelity polymerase Advantage.
2. We respectively combined R + L by fusion PCR.
Figure 2| R+L: The fusion PCR of 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-RL. Then we verified them using digestion and sequencing.
Figure 3| (1) The sample without restriction endonuclease (2) The vector was digested by PstI +XhoI.
4. We transferred the piGEM-RL into BL21 (DE3) and conducted the inducible expression. The color and concentration of bacterium liquid are as following:
Figure 4| The color and the concentration of bacterium liquid. (A) The color degree among different bacterium liquid. The higher concentration of bacterium liquid shows redder. (B)The OD600 of different concentration of bacterium liquid at different time points using ultraviolet spectrophotometer.
5. We transferred the piGEM-RL into BL21 (DE3) and conducted the inducible expression. The color and concentration of bacterium liquid are as following:
Figure 5| Testing expression of RFP + laccase in E.coli. M: marker. Lane 1: Untransformed bacteria. Lane 2: Bacteria which contain piGEM-RL. Induced at 37centigrades, 180rpm, and 10 hours with 0.5mM IPTG.
6. We transferred the piGEM-RL into BL21 (DE3) and conducted the inducible expression. The color and concentration of bacterium liquid are as following:
Figure 6| The color of supernate and activity of Laccase. (A)The higher concentration of laccase shows redder. (B)The activity of RFP + Laccase. Ultrasonic Cell Disruptor to crush the bacterium in ice-bath. We collected the Supernatant and detected the activity of laccase by ABTS method (http://2015.igem.org/Team:CHINA_CD_UESTC/Protocol). The 1mL supernate equal to the 5mL bacterium liquid which were cultivated at different time points.
Sequence and features BBa_K1779204 Sequence and features Not understood
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