Part:BBa_K2963032

racE- encoding racemase

Our project used this part to produce γ-PGA with different D/L glutamate monomer ratios. This part is constructed using tac promoter, racE gene and so on. We used plasmid PZM1 to construct it. This composite part is compared with the other part (BBa_K2963033) which was added another operator gene (lacO) to the tac promoter 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 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 lacO compared to one lacO. At the same time, we tested the enzyme activity of the racemase in cells. The enzyme activity of tac promoter with one lacO is higher than that with two lacO. The datas are shown as below.

Figure1

Figure 1. The expression level of the racE gene 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 lacO compared to one lacO.

Figure2

Figure 2. We tested the enzyme activity of the racemase in cells. The enzyme activity of tac promoter with one lacO is higher than that with two lacO.

References

1.Keitarou K. Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis[J]. Microbiology, 2004, 9(150): 2911-2920.

2. Feng Jiang, Gaofu Qi, Zhixia Ji. Expression of glr gene encoding glutamate racemase in Bacillus licheniformis WX-02 and its regulatory effects on synthesis of poly-γ-glutamic acid[J]. Biotechnology Letters, 2011, 33: 1837-1840.