Difference between revisions of "Part:BBa K2762003"

Revision as of 13:38, 17 October 2018

Phosphoribulokinase (prk)

Background

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).

Expression in E. coli

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 DH5α with and without plasmid carrying prk gene, each in separate glucose and xylose M9 mediums after a 12-hour incubation.

Sequence and Features

Source

Codon oprimized Synechococcus elongatus PCC7942.

Reference

Fuyu Gong1,2, Guoxia Liu1, Xiaoyun Zhai1,2, Jie Zhou1, Zhen Cai1* and Yin Li1 .(2015,Jun 18). Biotechnology for BiofuelsQuantitative analysis of an engineered CO₂-fixing Escherichia coli reveals great potential of heterotrophic CO₂ fixation