Part:BBa_K2963032

racE- encoding racemase

Our project used this part and part （BBa_K2963033）to produce γ-PGA with different D/L glutamate monomer ratios. This part contains the racE racemase gene, which was constructed using the tac (BBa_K864400) promoter, rbs (BBa_K2963006), racE gene (BBa_K2963004) and the T7 terminator (BBa_K2598024). The parts BBa_K864400, BBa_K2963006, BBa_K2598024 are contained in the ePathBrick loop vector pZM1 (Ptac) containing the inducible promoter Ptac. This composite part was compared with the other part (BBa_K2963033) which was added another operator gene (lacI) to the tac promoter（BBa_K864400） and the rest basic parts are the same. We used racemase activity data and real-time PCR data to characterize this composite part. 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 8h,16h,24h 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.