AmilCP-eCPX fusion with universal expression
Figure 1. Electrophoresis of inserts amilCP-eCPX fusion with universal expression from optimization gradient PCR of 25 µL reaction, resulting in 72°C as the optimal temperature for the annealing process with a DNA weight of 1373 bp and a thin other band of impurities. Nanodrop = 105.26 ng/µL
AmilCP-eCPX fusion with T7 expression
Figure 2. Electrophoresis of inserts amilCP-eCPX fusion with T7 expression from optimization gradient PCR of 25 µL reaction, resulting in 72°C as the optimal temperature for the annealing process with a DNA weight of 1386 bp and a thin other band of impurities. Nanodrop = 26 ng/µL
TEV protease-eCPX fusion with universal expression
Figure 3. Electrophoresis of inserts TEV protease-eCPX fusion with universal expression from optimization gradient PCR of 25 µL reaction, resulting in 62°C as the optimal temperature for the annealing process with a DNA weight of 1463 bp and a thin other band of impurities. Nanodrop = 53 ng/µL
TEV protease-eCPX fusion with T7 expression
Figure 4. Electrophoresis of inserts TEV protease-eCPX fusion with T7 expression from optimization gradient PCR of 25 µL reaction, resulting in 62°C as the optimal temperature for the annealing process with a DNA weight of 1449 bp and a thin other band of impurities. Nanodrop = 25 ng/µL
TEV protease through sec pathway (Universal & T7 systems)
Figure 5. Electrophoresis of inserts TEV protease through sec pathway represent 965 bp (160.588 ng/µL) as the result of universal expression construct and 951 bp (124.488 ng/µL) as the result of T7 expression construct. This result gets from optimization gradient PCR of 10 µL reaction, it also shows 72oC as the optimal temperature for the annealing process with a thin other band of impurities.
Cells transformation with amilCP-eCPX fusion (chloramphenicol antibiotic screening)
Figure 6. The results of the 33 µg/mL chloramphenicol antibiotic screening which had been diluted 10-5 to all the samples; Fig. 6A represents BL21 transformation with amilCP-eCPX fusion with universal expression (constitutive promoter); Fig. 6B. Represents Transformation BL21(DE3) with amilCP-eCPX fusion with T7 expression.
Cells transformation with TEV protease fusion (kanamycin antibiotic screening)
Figure 7. The results of the 30 µg/mL kanamycin antibiotic screening which had been diluted 10-5 to all the samples; Fig. 7A represents BL21 transformation with TEV protease-eCPX fusion with universal expression (constitutive promoter); Fig. 7B. Represents Transformation BL21(DE3) with TEV protease-eCPX fusion with T7 expression.
Cell transformation with TEV protease fusion sec pathway (kanamycin antibiotic screening)
Figure 8. The results of the 30 µg/mL kanamycin antibiotic screening which had been diluted 10-5 to all the samples; Fig. 8A represents BL21 transformation with TEV protease-eCPX fusion through sec pathway with universal expression (constitutive promoter); Fig. 8B. Represents Transformation BL21(DE3) with TEV protease-eCPX fusion through sec pathways with T7 expression.
The influence of incubation time on competent cell growth
Figure 9.The curve represents the effect of incubation time on the number of cells BL21 and BL21 (DE3). These data show that the incubation time is proportional to the increasing number of cells (the longer the incubation time, the more cells formed). The growth of E.coli BL21 (DE3) more faster compared to BL21 when viewed from the absorbance curve during incubation time at 180 minutes and 270 minutes, but the protein concentration produced by BL21 was higher than BL21 (DE3) base on figure 10.