Difference between revisions of "Part:BBa K2963039"
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | The BCA genes from Bacillus sp. encode a γ-PGA synthetase located on the cell membrane which is capable of polymerizing glutamic acid to form poly-γ-glutamic acid. In Bacillus licheniformis BCA are called capBCA. We | + | The <i>BCA</i> genes from <i>Bacillus sp.</i> encode a γ-PGA synthetase located on the cell membrane which is capable of polymerizing glutamic acid to form poly-γ-glutamic acid. In <i>Bacillus licheniformis</i>, the <i>BCA</i> are called <i>capBCA</i>. We used the <i>B* </i>gene a mutant of <i>B</i> gene. |
| − | The racE gene is derived from Bacillus subtilis and it encodes a racemase which can converts L-glutamate to D-glutamate. | + | The <i>racE</i> gene is derived from <i>Bacillus subtilis</i> and it encodes a racemase which can converts L-glutamate to D-glutamate. |
| − | We used this part to produce different D/L monomer ratios | + | We used this part to produce γ-PGA with different D/L monomer ratios. We assembled the <i>capB*CA</i> genes and the part containing <i>racE</i> gene (BBa_K2963032) together to construct this part using plasmid PZM1. |
===Characterization=== | ===Characterization=== | ||
| − | We transferred this part into our chassis microorganism Corynebacterium glutamicum | + | We transferred this part into our chassis microorganism <i>Corynebacterium glutamicum</i> and used HPLC to detect the D/L monomer ratio of γ-PGA.The result shows as below. |
| + | Picture 1 | ||
[[image:HPLC.png|400px]] | [[image:HPLC.png|400px]] | ||
| − | Picture1 is the L-glutamate monomer ratio in γ-PGA we have produced using part BBa_K2963009 and the result reaches about over 90%. | + | Picture1 is the L-glutamate monomer ratio in γ-PGA we have produced using part BBa_K2963009 which do not link with <i>racE</i> gene and the result reaches about over 90%. |
| − | + | ||
| + | Picture 2 | ||
[[image:HPLC3.png|400px]] | [[image:HPLC3.png|400px]] | ||
| − | Picture2 is the result of BBa_K2963039. This part contains capBCA genes and racE gene which is under the control of tac promoter with one lacO. The L-glutamate monomer ratio reaches about 32%. | + | Picture2 is the result of BBa_K2963039. This part contains <i>capBCA</i> genes and <i>racE</i> gene which is under the control of tac promoter with one <i>lacO</i>. The L-glutamate monomer ratio reaches about 32%. |
| + | The results show that when we linked <i>capB*CA</i> genes with <i>racE</i> gene, the L-glutamate monomer ratio of γ-PGA is changing. | ||
This part is working. All the results show that we have achieved the goal of producing different D/L glutamate monomer ratio of γ-PGA preliminary. | This part is working. All the results show that we have achieved the goal of producing different D/L glutamate monomer ratio of γ-PGA preliminary. | ||
| + | |||
| + | ===References=== | ||
| + | 1. Xu P, Vansiri A, Bhan N, et al. ePathBrick: a synthetic biology platform for engineering metabolic pathways in E. coli[J]. ACS Synthetic Biology, 2012, 1(7): 256-266. | ||
| + | |||
| + | 2.Mutalik, Vivek K, et al. "Precise and reliable gene expression via standard transcription and translation initiation elements." Nature Methods 10.4(2013):354. | ||
Latest revision as of 11:19, 20 October 2019
Producing γ-PGA with different D/L glutamate monomer ratio(R).
Usage and Biology
The BCA genes from Bacillus sp. encode a γ-PGA synthetase located on the cell membrane which is capable of polymerizing glutamic acid to form poly-γ-glutamic acid. In Bacillus licheniformis, the BCA are called capBCA. We used the B* gene a mutant of B gene. The racE gene is derived from Bacillus subtilis and it encodes a racemase which can converts L-glutamate to D-glutamate.
We used this part to produce γ-PGA with different D/L monomer ratios. We assembled the capB*CA genes and the part containing racE gene (BBa_K2963032) together to construct this part using plasmid PZM1.
Characterization
We transferred this part into our chassis microorganism Corynebacterium glutamicum and used HPLC to detect the D/L monomer ratio of γ-PGA.The result shows as below.
Picture 1
Picture1 is the L-glutamate monomer ratio in γ-PGA we have produced using part BBa_K2963009 which do not link with racE gene and the result reaches about over 90%.
Picture 2
Picture2 is the result of BBa_K2963039. This part contains capBCA genes and racE gene which is under the control of tac promoter with one lacO. The L-glutamate monomer ratio reaches about 32%.
The results show that when we linked capB*CA genes with racE gene, the L-glutamate monomer ratio of γ-PGA is changing. This part is working. All the results show that we have achieved the goal of producing different D/L glutamate monomer ratio of γ-PGA preliminary.
References
1. Xu P, Vansiri A, Bhan N, et al. ePathBrick: a synthetic biology platform for engineering metabolic pathways in E. coli[J]. ACS Synthetic Biology, 2012, 1(7): 256-266.
2.Mutalik, Vivek K, et al. "Precise and reliable gene expression via standard transcription and translation initiation elements." Nature Methods 10.4(2013):354.