For generating our Toolbox, we used variants of two different aminoacyl tRNA/synthetases (aminoacyl tRNA/RS),
tyrosyl and pyrrosylyl. When working with our toolbox, we recognized that the
Escherichia coli cells containing these different aminoacyl tRNA/RS plasmids had different characteristics. We could confirm this assumption by performing growth experiments with
E.coli transformed with our different aminoacyl tRNA/synthetases.
Therefore, we transformed the certain aminoacyl tRNA/RS plasmid and two controls (
pSB1C3 and
pSB3T5) in
E. coli BL21 (DE3). We performed the cultivation in 1 mL LB-media with the matching non canonical amino acid (ncAA) in 12‑well microtiter plates by 600 rpm at 37 °C. We used two biological replicates, each with three technical replicates of the optical density of each cultivation by NanoDrop.
When depicting the growth rate of the cells, containing the aminoacyl tRNA/RS on the high copy plasmid pSB1C3 (Figure 1), the difference in growth is striking. The growth experiments show that the incorporation of ncAAs through the amber codon are a major metabolic pressure for the cells, resulting in a 0.25 to 0.5 % slighter growth rate than of the control. Only the
PrK‑tRNA/synthetase showed a better growth of up to 17 % better than the control. This can be explained by the metabolic pressure of pSB1C3 by coding mRFP. In addition, the very specific incorporation of the ncAA by the PrK (Figure 3) also supports a faster growth of the culture, containing the PrK aminoacyl tRNA/synthetase. That is due to the similarity of the used ncAA Prk to the original amino acid pyrrolysyl, requiring only one point mutation of the aminoacyl synthetase (aaRS). This could be demonstrated by us with our CFP‑YFP system(
BBa_K2201343)for the efficiency of the incorporation of ncAA.
