Part ImprovementThis year, we have improved the previous part BBa_J23100 by adding RBS and pelB-5D to make it can be used for extracelluar expression. And the part number is BBa_K2617017. The combination of promoter, RBS and signal peptide makes it convenient to use as a Biobrick and gives it the function of extracellular expression.For the function determination, we chose the PETase as our reporter protein because its activity can be easily detected. Thus we constructed two plasmids with and without pelB-5D respectively to compare the function of our improved part and previous part.The construction of plasmids were as followed.Table 1 The introduction of Positive Control and Negative Control
No. Vector Vector map Description 1 Positive Control BBa_J23100-RBS-pelB+5D-PETase-Ter 2 Negative Control BBa_J23100-RBS-PETase-Ter
Experiment ResultFor quantitative assay, a standard curve of pNP ranging from 0-0.8 mM in 100 mM of phosphate buffer (pH 7.4) was measured(Fig. 1).Fig. 1 The standard curve of pNP.The activity of the extracelluar fractions was detected by the method of pNPB. And the results were as followed(Fig. 2).Fig. 2 The activity for extracellular fractions of Positive Control and Negative Control
ConclusionBased on our functional determination test results, the improvement that we did has succeeded. As you could see, PETase that carrying our improved part (Fig. 2) has much higher activity level than the PETase that carrying promoter BBa_J23100 but without pelB-5D. From our experiment result, we could conclude that our improved part indeed gives the promoter BBa_J23100 the function of extracellular expression. So, our team has successfully improve the part BBa_J23100 with the addition of RBS and pelB-5D.
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