Difference between revisions of "Part:BBa K622005:Experience"

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===Characterization of &sigma;<sup>54</sup> promoter activity===
 
===Characterization of &sigma;<sup>54</sup> promoter activity===
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We performed the function check of &sigma;<sup>54</sup> promoter in the genome of ''E. coli''.
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By measuring the amount of mRNA of glnG, the promoter activities in the different glutamine concentration were measured (Fig.1). The activity in 0.0 % glutamine was measured as a standard and those in 0.2 and 2.0 % glutamine were compared to the standard. Figure 1 shows that the activities in 0.2 and 2.0 % glutamine are repressed to less than one-fifth of that in 0.0 % glutamine.
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[[Image:Kyoto-sigma54-1.png|400px|thumb|center|Fig.1: The &sigma;<sup>54</sup> promoter activity in the different glutamine concentration. The activity in 0.0 % glutamine was measured as a standard and the activities in 0.2 and 2.0 % glutamine were the relative values.]]
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After the overnight cultivation in M9 medium with glucose and casamino acids, the medium was replaced with fresh M9 medium with glutamine not containing glucose or casamino acids and the incubation at 37 degrees was started. After the 13 minutes incubation, RNA was extracted, cDNA was synthesized, and real time PCR was performed.
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 17:55, 14 October 2011

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Characterization of σ54 promoter activity

We performed the function check of σ54 promoter in the genome of E. coli.

By measuring the amount of mRNA of glnG, the promoter activities in the different glutamine concentration were measured (Fig.1). The activity in 0.0 % glutamine was measured as a standard and those in 0.2 and 2.0 % glutamine were compared to the standard. Figure 1 shows that the activities in 0.2 and 2.0 % glutamine are repressed to less than one-fifth of that in 0.0 % glutamine.

Fig.1: The σ54 promoter activity in the different glutamine concentration. The activity in 0.0 % glutamine was measured as a standard and the activities in 0.2 and 2.0 % glutamine were the relative values.

After the overnight cultivation in M9 medium with glucose and casamino acids, the medium was replaced with fresh M9 medium with glutamine not containing glucose or casamino acids and the incubation at 37 degrees was started. After the 13 minutes incubation, RNA was extracted, cDNA was synthesized, and real time PCR was performed.

User Reviews

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