While most organisms utilize 20 canonical amino acid building blocks for protein synthesis, adding additional noncanonical amino acids (ncAAs) to the amino acid repertoire can greatly facilitate the investigation of protein structures and functions. Genetic incorporation of ncAAs requires 3 key components: a “blank” (normally non-coding) codon, a biorthogonal translational system and sufficient amount of intracellular ncAAs. Despite significant efforts focused on biorthogonal machinery and available codon for encoding ncAAs, current methodologies to reach sufficient amount of cellular ncAAs have largely relied on the exogenous feeding of chemically-synthesized ncAAs and successful uptake of these ncAAs by cells. The requirement of external addition of ncAAs limits the development of genetic code expansion in its efficiency as well as applicability in both academic and industrial settings. To solve this limitation, creation of completely autonomous cells with both biosynthetic pathway and genetic incorproation mechinery for ncAAs has been accomplished in this thesis. Before engineering cells with novel ncAA as its 21st amino acid, the reported bacterial cells with p-aminophenylalanine (pAF) as its 21st amino acid was optimized and its application to produce proteins with site-specific modifications was explored. Two bacterial cells with novel ncAAs (5-hydroxyltryptophan and 3,4-dihydroxylphenylalanine) as its 21st amino acid have been created by introducing external biosynthetic pathways reported in the literature and their corresponding genetic incorporation machineries. Comparing with classical genetic expansion technology, cells with those ncAAs as their 21st amino acid have been demonstrated with superior ability to detect oxidative stress and prepare site-specific conjugate of therapeutic proteins. Beyond the usage of reported biosynthetic way, a novel sulfotransferase was discovered from bioinformatics for biosynthesizing sulfotysoine (sTyr), which is inefficient to penetrate cell membrane. As an important protein post-translational modification, sTyr produced in situ could be genetically incorporated into proteins in both prokaryotic and eukaryotic cells with higher efficiency than classical genetic code expansion based on exogenous addition of sTyr. The cells with sTyr as its 21st amino acid was applied to produce thrombin inhibitors with enhanced affinity. As another component towards creation of cells with a 21st amino acid, the genetic incorporation machineries for two isocyanide-containing ncAAs were discovered and the genetic incorporation of this novel functional group was used for protein activation and site-specific conjugation.