Difference between revisions of "Part:BBa K3209000"
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==Experiment Results:== | ==Experiment Results:== | ||
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Figure 1. The synthesis of Glycolaldehyde and Dihydroxyacetone from formaldehyde, catalyzed by BFD-M7. This enzyme can catalyze the synthesis of two compounds Glycolaldehyde and Dihydroxyacetone, using formadehyde. | Figure 1. The synthesis of Glycolaldehyde and Dihydroxyacetone from formaldehyde, catalyzed by BFD-M7. This enzyme can catalyze the synthesis of two compounds Glycolaldehyde and Dihydroxyacetone, using formadehyde. | ||
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Figure 2. Identification of BDD-M7 and TalB-F187Y expression vectors construction. | Figure 2. Identification of BDD-M7 and TalB-F187Y expression vectors construction. | ||
1:BFD-M7 plasmid; 2:Digestion of BFD-M7 plasmid by EcoRI and PstI; 3:TalB-F187Y plasmid; 4:Digestion of TalB-F187Y plasmid by EcoRI and PstI; M: Marker. | 1:BFD-M7 plasmid; 2:Digestion of BFD-M7 plasmid by EcoRI and PstI; 3:TalB-F187Y plasmid; 4:Digestion of TalB-F187Y plasmid by EcoRI and PstI; M: Marker. | ||
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Figure 3. Expression of BFD-M7 and purification by Ni-NTA affinity chromatography. | Figure 3. Expression of BFD-M7 and purification by Ni-NTA affinity chromatography. | ||
M:protein marker; 1:precipitation samples in the cell lysates; 2:supernatant samples in the cell lysates; 3:50 mM imidazole eluent; 4:100 mM imidazole eluent; 5:200 mM imidazole eluent. This figure showed that 200mM imidazole eluent is the best concentration for elution expressed BFD-M7. | M:protein marker; 1:precipitation samples in the cell lysates; 2:supernatant samples in the cell lysates; 3:50 mM imidazole eluent; 4:100 mM imidazole eluent; 5:200 mM imidazole eluent. This figure showed that 200mM imidazole eluent is the best concentration for elution expressed BFD-M7. | ||
Revision as of 23:27, 20 October 2019
Benzoylformate decarboxylase mutant (BFD-M7)
This is benzoylformate decarboxylase mutant (BFD-M7) from Pseudomonas putida. It contains 7 amino acids mutations, which sequence is different from BFD (BBa_K2155001). The mutation information of BFD-M7 is as follows: W86R, N87T, L109G, L110E, H281V, Q282F and A460M. BFD-M7 can catalyze glycolaldehyde synthesis from formaldehye, then the glycolaldehyde reacts with formaldehyde to form dihydroxyacetone (DHA), also catalyzed by this enzyme.
Experiment Results:
Figure 1. The synthesis of Glycolaldehyde and Dihydroxyacetone from formaldehyde, catalyzed by BFD-M7. This enzyme can catalyze the synthesis of two compounds Glycolaldehyde and Dihydroxyacetone, using formadehyde.
Figure 2. Identification of BDD-M7 and TalB-F187Y expression vectors construction.
1:BFD-M7 plasmid; 2:Digestion of BFD-M7 plasmid by EcoRI and PstI; 3:TalB-F187Y plasmid; 4:Digestion of TalB-F187Y plasmid by EcoRI and PstI; M: Marker.
Figure 3. Expression of BFD-M7 and purification by Ni-NTA affinity chromatography.
M:protein marker; 1:precipitation samples in the cell lysates; 2:supernatant samples in the cell lysates; 3:50 mM imidazole eluent; 4:100 mM imidazole eluent; 5:200 mM imidazole eluent. This figure showed that 200mM imidazole eluent is the best concentration for elution expressed BFD-M7.
Figure 4. HPLC analysis of the products catalyzed by BFD-M7.
A: Standard glycolaldehyde; B: Standard DHA; C: The products catalyzed by BFD-M7 in vitro. The results showed that our expression vector expressed active BFD-M7, which catalyzed the synthesis of glycolaldehyde and DHA.
References:
- Bo Cui, Bingzhao Zhuo, Xiaoyun Lu, et al. Enzymatic synthesis of xylulose from formaldehyde. Chinese Journal of Biotechnology,2018, 34(7): 1128-1136.
- Xiaoyun Lu, Yuwan Liu, Yiqun Yang, et al. Constructing a synthetic pathway for acetyl
coenzyme A from one-carbon through enzyme design. Nature communications, 2019 Mar 26;10(1):1378.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 771
Illegal NgoMIV site found at 1233
Illegal AgeI site found at 235 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 340