Difference between revisions of "Part:BBa K2963033"
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This part contains the racE racemase gene, which was constructed using a modified tac promoter (BBa_K2963030) with another additional operator gene (lacI), rbs (BBa_K2963006), racE gene (BBa_K2963004) and the T7 terminator (BBa_K2598024). The parts BBa_K2963030, BBa_K2963006, BBa_K2598024 are contained in the ePathBrick loop vector pZM1 (Ptac) containing the inducible tac promoter. This part is compared with part (BBa_K2963032) and we used racemase activity data and real-time PCR data to characterize these two composite parts and compare the different expression intensities of racE gene using different structures of tac promoters. | This part contains the racE racemase gene, which was constructed using a modified tac promoter (BBa_K2963030) with another additional operator gene (lacI), rbs (BBa_K2963006), racE gene (BBa_K2963004) and the T7 terminator (BBa_K2598024). The parts BBa_K2963030, BBa_K2963006, BBa_K2598024 are contained in the ePathBrick loop vector pZM1 (Ptac) containing the inducible tac promoter. This part is compared with part (BBa_K2963032) and we used racemase activity data and real-time PCR data to characterize these two composite parts and compare the different expression intensities of racE gene using different structures of tac promoters. | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
Revision as of 14:50, 15 October 2019
racE- encoding racemase
Our project used this part and part （BBa_K2963032）to produce γ-PGA with different D/L glutamate monomer ratios.
This part contains the racE racemase gene, which was constructed using a modified tac promoter (BBa_K2963030) with another additional operator gene (lacI), rbs (BBa_K2963006), racE gene (BBa_K2963004) and the T7 terminator (BBa_K2598024). The parts BBa_K2963030, BBa_K2963006, BBa_K2598024 are contained in the ePathBrick loop vector pZM1 (Ptac) containing the inducible tac promoter. This part is compared with part (BBa_K2963032) and we used racemase activity data and real-time PCR data to characterize these two composite parts and compare the different expression intensities of racE gene using different structures of tac promoters.
Usage and Biology
The racE gene is derived from Bacillus subtilis. This gene encodes a racemase which can converts L-glutamic acid to D-glutamate.
Characterization
We used real-time quantitative PCR and enzyme activity assay to compare the different expressions of racE gene under the control of tac promoter containing different numbers of operator genes in Corynebacterium glutamicum. The results were shown in Figure 1. The expression level of the racE gene at 8 h, 16 h, 24 h decreased by 29.99%, 58.86% and 62.34% respectively. The results showed that the expression intensity of racE was effectively reduced at each transcriptional stage by the tandem of two lacOs compared to one lacO. At the same time, we tested the enzyme activity of the racemase in cells. The result was the same as the above and the enzyme activity was different. The data is shown in Figure 2.
Figure 1. The expression level of the racemase gene racE at 8 h, 16 h, 24 h decreased by 29.99%, 58.86% and 62.34% respectively. The results show that the expression intensity of racE was effectively reduced at each transcriptional stage by the tandem of two lacOs compared to one lacO.
Figure 2. we tested the enzyme activity of the racemase in cells. The result was the same as the above and the enzyme activity was different.