Difference between revisions of "Part:BBa K2963032"
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<partinfo>BBa_K2963032 short</partinfo> | <partinfo>BBa_K2963032 short</partinfo> | ||
| − | This part | + | Our project used this part to produce γ-PGA with different D/L glutamate monomer ratios. |
| + | This part is constructed using tac promoter, <i>racE</i> 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 (<i>lacO</i>) 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 <i>racE</i> gene using different tac promoters. | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | < | + | The <i>racE</i> gene is derived from <i>Bacillus subtilis</i>. 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 <i>racE</i> gene under the control of tac promoter containing different numbers of operator genes in <i>Corynebacterium glutamicum</i>. | ||
| + | The results were shown in Figure 1. The expression level of the <i>racE</i> 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 <i>racE</i> was effectively reduced at each transcriptional stage by the tandem of two <i>lacO</i> compared to one <i>lacO</i>. At the same time, we tested the enzyme activity of the racemase in cells. The enzyme activity of tac promoter with one <i>lacO</i> is higher than that with two <i>lacO</i>. The datas are shown as below. | ||
| + | |||
| + | Figure1 | ||
| + | [[image:Figure1-gaohaixin.png|400px]] | ||
| + | |||
| + | Figure 1. The expression level of the <i>racE</i> gene at 8h,16h,24h decreased by 29.99%, 58.86% and 62.34% respectively. The results show that the expression intensity of <i>racE </i> was effectively reduced at each transcriptional stage by the tandem of two <i>lacO</i> compared to one <i>lacO</i>. | ||
| + | |||
| + | Figure2 | ||
| + | [[image:Figure2-gaohaixin.png|400px]] | ||
| + | |||
| + | Figure 2. We tested the enzyme activity of the racemase in cells. The enzyme activity of tac promoter with one <i>lacO</i> is higher than that with two <i>lacO</i>. | ||
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| + | |||
| + | ===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. | ||
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| + | <!-- Add more about the biology of is part here. | ||
| − | |||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K2963032 parameters</partinfo> | <partinfo>BBa_K2963032 parameters</partinfo> | ||
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Latest revision as of 14:21, 19 October 2019
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.