Difference between revisions of "Part:BBa K2762003"
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==Characterization== | ==Characterization== | ||
===SDS-PAGE of PRK=== | ===SDS-PAGE of PRK=== | ||
| − | 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 | + | 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 O.D.600 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. | 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. | The result is shown below. | ||
Revision as of 08:11, 29 September 2018
Phosphoribulokinase (prk)
Usage and Biology
This part encodes for phosphoribulokinase (PRK) (EC 2.7.1.19), 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 O.D.600 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]