Difference between revisions of "Part:BBa K2762007"

Revision as of 09:09, 29 September 2018

P_lacI-B0034-prk-B0015

Usage and Biology

This composite part is composed of a consecutive promoter BBa_R0010, a strong ribosomal binding site BBa_B0034 and a PRK-encoding gene BBa_K2762003. 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 DH5α with and without plasmid carrying prk gene, each in separate glucose and xylose M9 mediums after a 12-hour incubation.

Sequence and Features