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

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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.
 
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.
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Experiment
 
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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.
 
  
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Result and Findings
  
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The competent cells of DH10B were prepared from the bacterial stock stored in the deep freezer in the lab.
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1.pH 4.5 is found to be the best pH that is suitable for the expression of Dendar2 .  
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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
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srarting culture was transferred into 1 ml LB medium with different pH respectively and continued to culture for 360 mins.  
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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.
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2.Thered fluorescence from Dendar2 after photoconversion showed stronger fluorescent intensity than before.
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ImageJ was used to analysize the florescent intensity of Dendar2 fluorescence.
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[[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. ]]
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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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[[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.]]
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[[File:Scau-china-2018-9.png|800px|thumb|center| Figure 3 The measurement of Dendar2, red fluorescence signal after photoconversion changed in different pH value gradients during 360 min.]]
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Result and Findings
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1.The acid condition is suitable for the expression of Dendar2 When pH =4.5, the part can have a better working condition .
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2.There red fluorescence we observed after photoconversion have stronger intensity than the green fluorescence before photoconversion.
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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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Latest revision as of 06:15, 16 October 2018


User Reviews

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.

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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