pet22b vector  (Addgene inc)

Journal: Nature Communications

Article Title: A robust platform streamlining aromatic noncanonical amino acid biosynthesis and genetic code expansion in Escherichia coli

doi: 10.1038/s41467-025-63679-6

Figure Lengend Snippet: a Transformation of p -iodobenzaldehyde (pIBzH) by various E. coli strains with reconstituted biosynthetic pathways was evaluated. The reaction activity of different E. coli strains for the conversion of pIBzH to pIF was assessed by supplementing LB cultures with 1 mM pIBzH at the time of expression induction. The products were analyzed using UPLC-MS. Conversions within 12 h were calculated. Error bars represent the mean ± s.d. of n = 3 independent biological samples. b Depiction of different plasmids for pIF synthesis and incorporation into sfGFP. pACYCDuet-1 contained threonine aldolase (PpLTA) and threonine deaminase (RpTD) genes for cascade catalysis, pCDF plasmid was used to express the GCE system for pIF encoding, and pET22b contained sfGFP bearing an amber mutation at Y151. Genes were expressed under IPTG-induction, except that tRNA was controlled by a constitutive promoter. c Production of sfGFP using pIF, biosynthesized from pIBzH, was carried out in E. coli BL21 (DE3) and RARE (DE3) hosts. The efficiency of production was evaluated based on the fluorescence intensity of sfGFP. The positive group was in the presence of 1 mM pIF (green bar), the experimental group was in the presence of 1 mM pIBzH (blue bar), and the negative group was without pIF or pIBzH (gray bar). Error bars represent the mean ± s.d. of n = 3 independent samples. d The effect of different reaction components on sfGFP production in E. coli RARE (DE3) carrying three plasmids was evaluated. In the negative control groups, one of the components was omitted, or no additional component was added, compared to the experimental group. Cultures were supplemented with 1 mM pIF or 1 mM pIBzH, and fluorescence was measured after 24 h of induction. The fluorescence intensity of cells cultured with 1 mM pIF was set as 100% for comparison. Error bars represent the mean ± s.d. of n = 3 independent samples. PG, positive group with 1 mM pIF; EG, experimental group with 1 mM pIBzH; NG1, negative group, PLP was omitted; NG2, negative group, Gly was omitted; NG3, negative group, pIBzH was omitted; NG4, negative group, without any components. e Mass characterization of sfGFP with pIF biosynthesized from pIBzH. The expected molecular mass (MW) value of sfGFP with pIF at Y151 was 27918 Da; observed MW value (as shown) was 27916 Da. Source data of ( a, c ) and ( d ) are provided in the Source Data file.

Article Snippet: Genes were codon-optimized, synthesized, and cloned into pET28a or pET22b expression plasmids by Genewiz.

Techniques: Transformation Assay, Activity Assay, Expressing, Plasmid Preparation, Mutagenesis, Fluorescence, Negative Control, Cell Culture, Comparison

Journal: Nature Communications

Article Title: A robust platform streamlining aromatic noncanonical amino acid biosynthesis and genetic code expansion in Escherichia coli

doi: 10.1038/s41467-025-63679-6

Figure Lengend Snippet: a Depiction of plasmids for ncAAs synthesis and incorporation into antibody fragments. Plasmids that expression of CsLTA and RpTD for ncAAs cascade catalysis and pAzFRS (2×)/tRNA Tyr pairs were same as Fig. . Mutants of four antibody fragments were constructed in the pET22b vector. pAzFRS (2×) was controlled by arabinose-controlled pBAD promoter, tRNA was controlled by constitutive promoter and other genes were expressed under IPTG-induction. b The yields of antibody fragments containing ncAAs (Mutant) after purification and comparison with wild type (WT). The cell was cultured with 1 mM pAzBzH, 50 mM Gly and 20 μM PLP and induced by arabinose and IPTG for 24 h. The proteins were purified with Ni-NTA column. c High-resolution mass of purified antibody fragments with pAzF (The high-resolution mass spectrometry of wild type antibody fragments were shown in Supplementary Fig. ). The purification experiments were repeated one time with result. d Confocal images of Her2-positive SK-Br-3 cells and Her2-negative MDA-MB-468 cells stained with anti-Her2-Fab-A121pAzF-AF488 conjugate. In consistent with literature (Ref. ), Her2-negative MDA-MB-468 cells was exploited as negative control in the fluorescence assay rather than wild type antibody because the click reaction between azide and alkyne group is commonly known as classic biorthogonal chemistry. Scale bars = 10 µm. Assays were repeated one time with result. Source data of ( b ) are provided in Source Data file.

Article Snippet: Genes were codon-optimized, synthesized, and cloned into pET28a or pET22b expression plasmids by Genewiz.

Techniques: Expressing, Construct, Plasmid Preparation, Mutagenesis, Purification, Comparison, Cell Culture, Mass Spectrometry, Staining, Negative Control, Fluorescence