Our Favorite Parts
In enzymology, a benzoylformate decarboxylase (EC 220.127.116.11) is an enzyme that catalyzes the following chemical reaction:
benzoylformate + H+ ⇌ benzaldehyde + CO2
However, in our project we used an engineered benzoylformate decarboxylase, which could catalyze formaldehyde to synthesize Glycolaldehyde and Dihydroxyacetone(Figure1 and Figure2). If you use it to catalyze reaction in vivo, It needs one cofactor, thiamin diphosphate.
Figure 1. DHA and Glycoaldchyde could be synthesize from Formaldehyde by BFD.
Figure 2. HPLC analysis of samples from in vitro experiment of BFD.
This year we fused BFD with CtDock, and then CtDock could bind with CtCohesin(Figure3), which is part of our scaffold protein. Therefore, our core enzyme could form a enzyme complex, and the catalytic efficiency could be greatly improved.
Figure 3. BFD is fused with dockerin and then it can bind with matching CtCohesin.
Transaldolase B is important for the balance of metabolites in the pentose-phosphate pathway. It can catalyze this reaction:
Sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate = D-erythrose 4-phosphate + D-fructose 6-phosphate
In our project, we use this enzyme to condense Glycolaldehyde and Dihydroxyacetone into D-xylulose(Figure 4 and Figure 5). If you use it to catalyze reaction in vivo, It needs one cofactor, thiamin diphosphate. TalB was cloned into the plasmid vector pET22b+ for expression. We purify this protein by His tag. This year we fused TalB with CcsDock, and then CcsDock could bind with CcsCohesin(Figure6), which is part of our scaffold protein. Therefore, our core enzyme could form a enzyme complex, and the catalytic efficiency could be
Figure 4. D-xylulose was synthesized from glycolaldehyde and DHA by TalB.
Figure 5. HPLC analysis of samples from in vitro experiment of TalB
Figure 6. TalB is fused with dockerin and then it can bind with matching CcsCohesin.
The Parts We Improved
This year, we reduced 27bp of a registered part(BBa_K1830003, 12bp at 5’ end and 15bp at 3’ end) and then created our new part CBM3b(BBa_K2155005). According to a paper published by Stéphanie Perret in 2013, Both modules(CBM3a and CBM3b) bind to phosphoric acid swollen cellulose (PASC) with the same affinity. So we use a smaller module(CBM3b) to achieve the same function in our project.
In scaffold protein, there is a linker between adjacent cohesin, and it is the linker’s length that determines the adjacent enzyme’s distance and influences the passing on of intermediates between co-located enzymes. Therefore, the linker’s length is a main influence of multi-enzyme system’s reaction efficiency. Based on a registered part(K1830004) submitted by iGEM15_Concordia’s, we isolated the cohesin module of this part from the linker, which makes following team could adjust linker’s length according to their requirement when they use this cohesin. We created a new part number for the cohesin module(BBa_K2155006). Besides, we applied this part in our scaffold protein(BBa_K2155012) with other cohesins, and proved it was functional.The details were documented in our Result Page.
BBa_K2155001 is a modified BFD enzyme that can condense formaldehyde into two intermediate products, dihydroxyacetone and hydroxyacetaldehyde.And B is a modified TalB enzyme that can condense these two intermediates into D-xyxulose.BBa_K2155013 is a combination of these two enzymes，so it can condense formaldehyde into D-xyxulose.BBa_K2155002, BBa_K2155004, BBa_K2155005, BBa_K2155006, BBa_K2155007, BBa_K2155008, BBa_K2155009, These parts can be combined into a self-assembled protein scaffold.Using this protein scaffold, the efficiency of the D-xylulose synthesis reaction can be increased.
|BBa_K2155001||BFD||Coding||Carbon chain elongation||Bingzhao Zhuo, Yue Yang||1584|
|BBa_K2155002||CtDock||Coding||Dockerin||Xin Yin, Bingzhao Zhuo||288|
|BBa_K2155003||TalB||Coding||Carbon chain elongation||Bingzhao Zhuo, Yue Yang||951|
|BBa_K2155004||CcsDockx||Coding||Dockerin||Yue Yang, Zexun Wu||261|
|BBa_K2155005||CBM3b||Coding||Dockerin||Yun Liu, Zexun Wu||477|
|BBa_K2155006||CtCohesin||Coding||Cellulose Binding Domain||BingZhao Zhuo, Yue Yang||402|
|BBa_K2155007||CcsCohesin||Coding||Cohesin||Yue Yang, Xin Hu||360|
|BBa_K2155008||RfCohesin||Coding||Cohesin||Xin Yin, Zexun Wu||378|
|BBa_K2155009||RfDock||Coding||Cohesin||Chenyuan Li, Zexun Wu||246|
|BBa_K2155010||BFD-CtDock||Coding||Dockerin||BingZhao Zhuo, Yue Yang, Zexun Wu||1880|
|BBa_K2155011||TalB-CcsDock||Coding||Fused protein||Yue Yang, Xin Hu, Bingzhao Zhuo||1220|
|BBa_K2155012||Scaffold||Coding||Fused protein||Xin Yin, Zexun Wu, Bingzhao Zhuo, Yue Yang, Yun liu||1641|
|BBa_K2155013||BTT||Coding||Synthetic Protein Scaffold||BingZhao Zhuo, Yue Yang||2176|