Difference between revisions of "Part:BBa K2762003"

 
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===Usage and Biology===
 
===Usage and Biology===
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Phosphoribulokinase (PRK) (EC 2.7.1.19) is an enzyme involved in the Calvin-Benson-Bassham (CBB) cycle that catalyzes the conversion of ribulose-5-phosphate (Ru5P) to ribulose-1,5-biphosphate (RuBP).
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==Characterization==
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===SDS-PAGE of PRK===
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To find out whether the gene <i>prk</i> is successfully expressed in <i>E. coli</i>, we conducted a SDS-PAGE test. The cells were harvested by centrifuging at 10,000×g for 10 min., and then washed with deionized water for 2 times. The cell density was adjusted to an OD600 of 5 as the sample of whole cell (WC, whole cell catalyst).
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Finally, WC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% separating gel and 4% stacking gel. Proteins were visualized by staining with Coomassie blue R-250 and were scanned with an Image scanner.
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The result is shown below.
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===PRK Toxicity Test===
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RuBP produced from the PRK catalyzed reaction cannot be metabolized by <i>E. coli</i> and the accumulation of RuBP in <i>E. coli</i> would cause cell growth arrest. To examine the relationship between the expression of PRK in <i>E. coli</i> and its growth, we carried out an experiment to measure the growth of <i>E. coli</i> strain DH5alpha with and without plasmid carrying <i>prk</i> gene, each in separate glucose and xylose M9 mediums after a 12-hour incubation.
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Revision as of 06:55, 29 September 2018


Phosphoribulokinase (prk)


Usage and Biology

Phosphoribulokinase (PRK) (EC 2.7.1.19) is an enzyme involved in the Calvin-Benson-Bassham (CBB) cycle that catalyzes the conversion of ribulose-5-phosphate (Ru5P) to ribulose-1,5-biphosphate (RuBP).

Characterization

SDS-PAGE of PRK

To find out whether the gene prk is successfully expressed in E. coli, we conducted a SDS-PAGE test. The cells were harvested by centrifuging at 10,000×g for 10 min., and then washed with deionized water for 2 times. The cell density was adjusted to an OD600 of 5 as the sample of whole cell (WC, whole cell catalyst). Finally, WC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% separating gel and 4% stacking gel. Proteins were visualized by staining with Coomassie blue R-250 and were scanned with an Image scanner. The result is shown below.

PRK Toxicity Test

RuBP produced from the PRK catalyzed reaction cannot be metabolized by E. coli and the accumulation of RuBP in E. coli would cause cell growth arrest. To examine the relationship between the expression of PRK in E. coli and its growth, we carried out an experiment to measure the growth of E. coli strain DH5alpha with and without plasmid carrying prk gene, each in separate glucose and xylose M9 mediums after a 12-hour incubation.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]