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Here, we developed a method of double incorporation of modified amino acids and fluorescent non-natural amino acids in a quantitative, position-specific manner to obtain modified and fluorescently labeled proteins.
Remarkably, the l-Glu γ-methyl ester (P7) efficiently substituted for methionine in all steps of translation; fully formylated peptides containing the expected double incorporation of P7 were observed in experiments with template e.
The results from the single incorporation hexitol scaffold isoCMe follow generally the pattern of the deoxyribose forms but with lower responses, with the exception of the double incorporation oligomer (T*GC CAT*), which is the only oligomer of those tested where there is apparently higher recognition of the hexitol scaffold relative to deoxyribose.
Interpretation of this result against the single incorporations T*GC and TGC* and the double incorporation TGC* C*AT suggest that even if the h- and d-isoCMe nucleosides are mainly base-pairing G (thus being recognised as C) the presence of two adjacent molecules will corrupt this recognition.
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It would seem that double incorporations are terminal in this system, and are not able to be recognised correctly to permit survival as both T and C residues.
The d-isoCMe nucleoside appears to be recognised mostly as a C and then T, agreeing with the previous in vitro data showing the base pairing of this nucleoside with G most strongly, although in both double incorporations (TGC* C*AT and T*GC CAT*) there was virtually no recognition even as a C (for pairing with G).
Tripling AA availability at 5 mU/l insulin doubled incorporation of [1-C]leucine [i.e., muscle protein synthesis (MPS), P < 0.01] without affecting the rate of leg protein breakdown (LPB; appearance of d5-phenylalanine).
Cells were treated by double thymidine incorporation 24 hours after transfection when required.
Corals with type C1 symbionts had 87% higher rETRmax which correlated with more than double 14C incorporation rates.
The products of Therminator-catalyzed, single and double glyceronucleotide incorporation were characterized by MALDI-TOF MS. The expected mass difference between a primer extended with one deoxyribonucleotide vs. one glyceronucleotide is 42 Daltons.
Given the complexity of our data when trying to incorporate five NSAAs per sfGFP, we chose to focus on only single and double pPaF incorporation.
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