Difference between revisions of "Part:BBa K515107:Experience"

Revision as of 08:33, 15 October 2018

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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 fluorescence signal of Dendar2 in t=100min and t=300min time point, pH=4.5 and pH=9.1.The part work well in DH10B and the we found that the alkaline condition have a negative influence on the expression of Dendar2 in DH10B.

Figure 2 The measurement of Dendar2, green fluorescence signal before photoconversion changed in different pH value gradients during 360 min.

Figure 3 The measurement of Dendar2, red fluorescence signal after photoconversion changed in different pH value gradients during 360 min.

Result and Findings 1.The acid condition is suitable for the expression of Dendar2 When pH =4.5, the part can have a better working condition . 2.There red fluorescence we observed after photoconversion have stronger intensity than the green fluorescence before photoconversion. 3.Compared with the characterisation show before in the DH5α stain, we made conclusion the expression of Dendar2 can be well in those two E. coli strain.|

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-The main goal of this characterisation was to find out which pH value condition was the most effective among 6 level pH value to obtain a suitable culture condition for the expression of Dendar2.We also found that the part which expressed the Dendar2 can work well in E.coli strain DH10B, and just like in the DH5 alpha mentioned in main page.
+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
-We wanted to test 6 pH value gradients that easily used in lab : 4.5/5.8/6.2/7.2/8,2/9,1, to see, in a 360 min culturing time, which one would be the best pH value culture condition for the DH10B to growth and express the Dendar2.
+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.
-We used the bacterial stock of our own, coming from commercial bacteria cultured and made competent using our protocol a few weeks before the experiment and conserved at -80°C.
-We made an electric shock transformation of 30ng BBa_K515107 part into 50μL bacteria and culture in LB plate, then picked the colony to 250μL LB broth medium for one hour. We prepared different pH level LB medium in 6 gradients(4.5/5.8/6.2/7.2/8,2/9,1), and add 25μL cultured medium from the 250μL LB broth medium to 1 mL different pH level LB medium, still culture to 360 min.
-Very 20 min time point, we collect 10μL bacterial culture medium and measure the green fluorescence as well as the red fluorescence after photoconversion using photon stimulation at UV wavelength.
+The competent cells of DH10B were prepared from the bacterial stock stored in the deep freezer in the lab.
-ImageJ was used to analysize the intensity of Dendar2 fluorescence.
+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.
 [[File:Scau-china-2018-7.png|800px|thumb|center| Figure 1 The fluorescence signal of Dendar2 in t=100min and t=300min time point, pH=4.5 and pH=9.1.The part work well in DH10B and the we found that the alkaline condition have a negative influence on the expression of Dendar2 in DH10B. ]]
 [[File:Scau-china-2018-8.png|800px|thumb|center|Figure 2 The measurement of Dendar2, green fluorescence signal before photoconversion changed in different pH value gradients during 360 min.]]