Part:BBa_K2963033:Design
Ptac(two lacOs)-racE- Producing γ-PGA with different D/L monomer ratio
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 971
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The strength of racE gene expression affects the proportion of D/L glutamate monomer in γ-PGA. In our project, we wanted to regulate the expression of the racE gene to produce γ-PGA with different D/L-glutamate monomer ratios. Tac Promoter contains a kind of operator gene called lacO. We changed the number of operator gene in tac promoter. We used this part to produce γ-PGA with different D/L glutamic acid monomer ratios. Analysis of the results of the preliminary fermentation experiments show that we can roughly produce γ-PGA containing different ratios of D/L glutamic acid monomers. More detailed experimental steps and experimental results please visit our experiment page.
Determination of glutamate racemase activity: The cells ferment for 24 h, and the fermentation broth is centrifuged at 4°C. Collect the cells at 10000 rpm. Wash and centrifuge the cells using 0.85% physiological saline. Repeat the wash 3 times. The cells are suspended in p H 8.0, 0.1 M Tris-HCl buffer. Sonicate for 10 min in an ice bath. Centrifuge at 12000 rpm for 30 min, and the supernatant (crude enzyme solution) is taken for immediate determination of the relevant enzyme activity. Using 100 μL of crude enzyme solution reacts with 100 μL of substrate (0.5 g/L L-glutamic acid) at 30 ° C for 30 min and the reaction is terminated by boiling water bath. The content of D-glutamic acid in the reaction solution is determined by a high performance liquid phase method.
Source
racE from Bacillus Genome.