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RESULTS

Ⅰ. Enzyme and protein assays

1. IPTG Gradient Induction

We found that the concentration of IPTG could have an effect on the expression of our circuits, in order to make a better expression of the protein in the cell, we decided to make a IPTG gradient induction for our gene circuits. After measuring the enzyme activity and ran SDS-PAGE, We found the optimal IPTG concentration . As shown in the graph, the enzyme activity data and SDS-PAGE show the most suitable concentration of the enzyme

Figure 1.1 SDS-PAGE Analysis of cell-free crude extracts LeuDH (34+L) from E.coli BL21. The arrow points to the optimal band of LeuDH

Figure 1.2 Different concentrations of IPTG on the Enzyme activity of 34+L

Figure 1.3 SDS-PAGE Analysis of cell-free crude extracts FDH (34+F) from E.coli BL21. The arrow points to the optimal band of FDH

Figure 1.4 Different concentrations of IPTG on the Enzyme activity of 34+F

Figure 1.5SDS-PAGE Analysis of cell-free crude extracts LeuDH (30+F) from E.coli BL21. The arrow points to the band of LeuDH

Figure 1.6 Different concentrations of IPTG on the Enzyme activity of 30+L

Figure 1.7 SDS-PAGE Analysis of cell-free crude extracts LeuDH (32+F) from E.coli BL21. The arrow points to the band of LeuDH.

Figure 1.8 Different concentrations of IPTG on the Enzyme activity of 32+L

Figure 1.9 SDS-PAGE Analysis of cell-free crude extracts LeuDH(30+L) and FDH(34+F) from E.coli BL21. The black arrow points to the band of FDH. The red black arrow points to the band of LeuDH.

Figure 1.10 Different concentrations of IPTG on the Enzyme activity of 30+L+34+

Figure 1.11 SDS-PAGE Analysis of cell-free crude extracts LeuDH (34+L)and FDH (34+L)from E.coli BL21. The black arrow points to the band of FDH. The red black arrow points to the band of LeuDH.

Figure 1.12 Different concentrations of IPTG on the Enzyme activity of 34+L+34+F

Figure 1.13 SDS-PAGE Analysis of cell-free crude extracts LeuDH(32+L) and FDH(34+F) from E.coli BL21. The black arrow points to the band of FDH. The red black arrow points to the band of LeuDH.

Figure 1.14 Different concentrations of IPTG on the Enzyme activity of 32+L+34+F

2. HPLC

Under the conditions of optimum concentration, we carried out the next step of catalysis and induction, Apart from taking all three whole circuits into analysis, we detected circuits with LeuDH or FDH separately.

Figure 2.1 The conversion rate of isolated connection

Figure 2.2 The conversion rate of series connection

No matter which kind of RBS, the conversion rate does not show too much of change compared with each other, indicating that RBS control would not make an effect if the whole system is expressed separately. Obviously, the conversion rate of series connected circuits is much higher than the other. As we mentioned on the whole-cell biocatalyst part, our project which make two genes into series connection would largely promote the productivity and conversion rate of L-tert Leucine.we found RBS_B0030 make the best performance among these three kinds of RBSs which indicate that the most suitable strength of RBS is located at this range.

Figure 2.3 The e.e.value of series connection

Enantiomeric excess (e.e.) value is a measurement of purity used for chiral substances. It reflects the degree to which a sample contains one enantiomer in greater amounts than the other. A racemic mixture has an e.e. value of 0%, while a single completely pure enantiomer has an e.e. value of 100%. A sample with 70% of one enantiomer and 30% of the other has an ee of 40%. The HPLC results also show the ideal e.e. value of our products. No matter which circuits of our project, the e.e. value all turns to be 99% which means the perfect optical purity of L-tert-leucine.