Generating our Toolbox, we used variants of two different aminoacyl tRNA/synthetases (aminoacyl tRNA/RS), namely
tyrosyl and pyrrosylyl. While apllying our toolbox, we recognized that the
Escherichia coli cells containing different aminoacyl tRNA/RS plasmids had different characteristics. We could further confirm this assumption by performing growth experiments with
E.coli that prior to the cultivation were transformed with our different aminoacyl tRNA/synthetases.
For this purpose, we transformed chosen aminoacyl tRNA/RS plasmid and two control vectors,
pSB1C3 and
pSB3T5, into
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. The optical density of each cultivation was measured with NanoDrop at 600 nm. The comparison of the growth rates of each cultivation for the cells containing the aminoacyl tRNA/RS in high copy plasmid pSB1C3, as depicted in
Figure 1, showed significant differences between different the cultures.
Additionally, the growth experiments show that the incorporation of ncAAs through the amber codon cause a major metabolic burden for the cells, resulting in a growth rate decreased by 0.25 and 0.5 %, as compared to control cultures. Only the
Propargyllysine (PrK)‑tRNA/synthetase showed growth of up to 17 % higher than the growth of the control cultures. This can be explained by the metabolic pressure, exerted by the pSB1C3, due to coding the mRFP. In addition, the highly specific incorporation of the ncAA by the PrK
(Figure 3) also supports faster growth of the culture, containing the PrK aminoacyl tRNA/synthetase. This is due to the similarity of the used ncAA Prk-couple to the wild type amino acid pyrrolysyl, requiring only one point mutation of the aminoacyl synthetase (aaRS). We could demonstrate this with our CFP‑YFP system(
BBa_K2201343)for the efficiency of the incorporation of ncAA.
