Part:BBa_K515107:Experience

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SCAU-China

The main goal of this characterization is to find out which pH value is the most effective among the 6 pH values in order to obtain a suitable culture condition for the expression of Dendar2.We further found that the part which expressed the protein Dendar2 worked well in E.coli strain DH10B, like in the DH5 alpha. Experiment To figure out the best cultural pH for protein expression, we have tested 6 different sets of pH that including: 4.5, 5.8, 6.2, 7.2, 8.2, 9.1, We also performed a time-lapse culture for 360 mins to find out the best time point for harvesting DH10B that expresses Dendar2.

The competent cells of DH10B were prepared from the bacterial stock stored in the deep freezer in the lab. 30 ng DNA of BBa_K515107 part was transformed into 50 μl DH10B competent cells by electroporation. The bacteria colonies growing on the selective LB plates were picked and cultured in 250 μl LB broth medium for one hour as the starting culture. Thereafter, 25 μl of the srarting culture was transferred into 1 ml LB medium with different pH respectively and continued to culture for 360 mins.

Every 20 mins, we collected 10 μl bacterial culture and measure the green fluorescence and the red fluorescence before and after photoconversion of Dendar2 by using photon stimulation under UV light. ImageJ was used to analysize the florescent intensity of Dendar2 fluorescence.

Figure 1 The fluorescent signal of Dendar2 in the time point of 100 min and 300 min, pH 4.5 and pH 9.1.The protein expression of the part works well in DH10B. In addition, we found that the alkaline condition have a negative impact on the expression of Dendar2 in DH10B.

Figure 2 Measurement of the signal intensity of green fluorescence from Dendar2, before photoconversion under different pH conditions within 360 min.

Figure 3 Measurement of the signal intensity of red fluorescence from Dendar2, after photoconversion under different pH conditions within 360 min.

Result and Findings

1.pH 4.5 is found to be the best pH that is suitable for the expression of Dendar2 .

2.Thered fluorescence from Dendar2 after photoconversion showed stronger fluorescent intensity than before.

3.Compared with the the expression pattern of Dendar2 as showed previously in DH5α, we concluded that the expression of Dendar2 works in both E.coli strains.

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