Difference between revisions of "Part:BBa K2963032"
 
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<partinfo>BBa_K2963032 short</partinfo>
 
<partinfo>BBa_K2963032 short</partinfo>
  
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Our project used this part and part （BBa_K2963033）to produce γ-PGA with different D/L glutamate monomer ratios.  
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Our project used this part to produce γ-PGA with different D/L glutamate monomer ratios.  
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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.
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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.
  
 
===Usage and Biology===
 
===Usage and Biology===
  
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The racE gene is derived from Bacillus subtilis. This gene encodes a racemase which can converts L-glutamic acid to D-glutamate.
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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===
 
===Characterization===
  
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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.
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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>.
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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.
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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.
  
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Figure1
 
[[image:Figure1-gaohaixin.png|400px]]
 
[[image:Figure1-gaohaixin.png|400px]]
  
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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.
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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>.
  
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Figure2
 
[[image:Figure2-gaohaixin.png|400px]]
 
[[image:Figure2-gaohaixin.png|400px]]
  
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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.
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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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Design：
 
  
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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.
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===References===
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Tac Promoter contains a kind of operator gene called lacO. We changed the number of operator gene in tac promoter assembling other basic parts to form our composite parts: BBa_K2963032 and BBa_K2963033. We assembled these two parts with our favorite part BBa_K2963009 to produce different D/L glutamic acid monomer ratios. Analysis of the results of the preliminary fermentation experiments showed that we can roughly produce γ-PGA containing different ratios of D/L glutamic acid monomers. Detailed experimental steps and experimental results please visit our EXPERIMENT page
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1.Keitarou K. Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis[J]. Microbiology, 2004, 9(150): 2911-2920.
  
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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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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 Figure1-gaohaixin.png

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 Figure2-gaohaixin.png

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.