Journal: The Journal of Biological Chemistry

Article Title: Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depleted Escherichia coli Host Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation *

doi: 10.1074/jbc.M117.776310

Figure Lengend Snippet: Plasmids and E. coli strains used in this study

Article Snippet: The RF1-depleted E. coli strain C321.ΔA strain developed by others ( 39 ) and used here for selenoprotein production was obtained from Addgene (catalogue no. 488998).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depleted Escherichia coli Host Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation *

doi: 10.1074/jbc.M117.776310

Figure Lengend Snippet: Production of Sec-containing TrxR1 using UAG for Sec validated with mass spectrometry. Utilizing expression with UAG targeting in C321.ΔA host cells, the purified enzymes were analyzed with electrospray mass spectrometry. These analyses revealed that rat TrxR1 (top) as well as human TrxR1 (bottom) were purified as dominant forms of intact Sec-containing enzymes (green arrows) and with very little UAG-truncated protein detected (red arrows). The x axes indicate the mass range analyzed.

Article Snippet: The RF1-depleted E. coli strain C321.ΔA strain developed by others ( 39 ) and used here for selenoprotein production was obtained from Addgene (catalogue no. 488998).

Techniques: Mass Spectrometry, Expressing, Purification

Journal: The Journal of Biological Chemistry

Article Title: Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depleted Escherichia coli Host Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation *

doi: 10.1074/jbc.M117.776310

Figure Lengend Snippet: Enzymatically active TrxR1 can be produced in E. coli without use of a SECIS element. Rat TrxR1 was purified from C321.ΔA host cells harboring the pABC2-rTRUAG plasmid with a UAG codon for Sec but lacking an engineered SECIS element. Using 45 nm TrxR1 produced with this approach, its activity was measured with a thioredoxin-linked insulin reduction assay, in which consumption of NADPH was followed as a decrease in absorbance at 340 nm (red curve). As reduced insulin is accumulated it precipitates, explaining increased A340 at later time points. As comparison, activities were also determined using equal concentrations (45 nm) of purified TrxR1 variants having a Sec-to-Cys substitution (U498C, blue curve) or Sec-to-Ser substitution (U498S, black curve). Calculated turnover numbers were 241.3, 16.8, and 2.6 min−1 for the three TrxR1 species, respectively. This should be compared with turnover of fully Sec-containing rat TrxR1 (Fig. 2, top panel) calculated to be 3568 min−1.

Article Snippet: The RF1-depleted E. coli strain C321.ΔA strain developed by others ( 39 ) and used here for selenoprotein production was obtained from Addgene (catalogue no. 488998).

Techniques: Produced, Purification, Plasmid Preparation, Activity Assay, Insulin Reduction Assay

Journal: The Journal of Biological Chemistry

Article Title: Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depleted Escherichia coli Host Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation *

doi: 10.1074/jbc.M117.776310

Figure Lengend Snippet: TrxR1 produced using UAG for Sec in absence of both RF1 and a SECIS element resulted in a mixture of Sec-containing enzyme, UAG-truncated variant, and additional forms presumed to be Sec-to-Gln or Sec-to-Lys substituted variants. Shown is an electrospray mass spectrometry analysis of rat TrxR1 produced using the pABC2-rTRUAG plasmid in C321.ΔA host cells. The three major species of enzyme detected corresponded in size to a native Sec-containing variant (green arrow), UAG-truncated enzyme (red arrow), and a variant likely corresponding to Sec-to-Gln- or Sec-to-Lys-substituted forms (blue arrow). See text for further discussion.

Article Snippet: The RF1-depleted E. coli strain C321.ΔA strain developed by others ( 39 ) and used here for selenoprotein production was obtained from Addgene (catalogue no. 488998).

Techniques: Produced, Variant Assay, Mass Spectrometry, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depleted Escherichia coli Host Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation *

doi: 10.1074/jbc.M117.776310

Figure Lengend Snippet: Production of enzymatically active human GPx1 and validation of its active site tetrad. A, enzymatic activity of recombinant human GPx1 (1 μm, circles) was assessed through the decrease of absorbance at 340 nm over time upon addition of H2O2 (0.25 mm) to a cuvette containing GR (1 unit/ml), GSH (1 mm), and NADPH (0.2 mm). Control reactions were either without hGPx1 (triangles) or without H2O2 (squares). B, electrospray mass spectrometry analysis of human GPx1 produced in C321.ΔA host cells was performed. The major peaks are annotated according to the enzyme species as inferred from their molecular weights, including a combination of Sec-to-Gln- and Sec-to-Lys-substituted variants (U49Q, U49K), a Sec-containing wild type GPx1 form (WT), as well as potentially oxidized forms of wild type GPx1 (WT+16 and WT+32). C, results of LC/MS mass spectrometry analyses of tryptic digests of the GPx1 preparation used in B are shown, confirming the existence of a Sec-containing peptide (red curve), a Sec-to-Gln peptide (blue curve), and a Sec-to-Lys peptide (black curve; Lys serves as a cleavage site for trypsin, hence resulting in a shorter peptide in this analysis). The sequences of the peptides are also indicated, and their ratios in percentages as indicated in parentheses were calculated from the area under the curve. D, similar assay was performed as shown in A, using either the wild type GPx1 preparation (circles) or the Q84A, W162A, and N163A variants (each at 1 μm), as indicated.

Article Snippet: The RF1-depleted E. coli strain C321.ΔA strain developed by others ( 39 ) and used here for selenoprotein production was obtained from Addgene (catalogue no. 488998).

Techniques: Activity Assay, Recombinant, Mass Spectrometry, Produced, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depleted Escherichia coli Host Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation *

doi: 10.1074/jbc.M117.776310

Figure Lengend Snippet: Schematic illustration of constructs, efficiencies, and yields from different systems of recombinant selenoprotein production as studied here. The schemes graphically summarize the principles for recombinant selenoprotein production using different E. coli host strains with alternative codon-anticodon combinations with or without RF1 and RF2, in the presence or absence of an engineered SECIS element, as discussed in the text and indicated in the figures. Shown are also the approximate final yields of selenoproteins purified from the different conditions, with Sec-containing variants shown in green, truncated in red, and suppression of the Sec codon with other amino acids such as Gln or Lys indicated in blue. A, expression of TrxR1 in BL21(DE3) with an engineered SECIS element and UGA for Sec is shown. B, results of the same expression of TrxR1 as in A is shown, but using UAG as the Sec codon together with a corresponding mutation of the SelC anticodon. C, results are shown using the same principle as in B, but changing from BL21(DE3) to the RF1-depleted C321.ΔA host strain. D, results producing TrxR1 or GPx1 are summarized, as obtained using the principle given in C, but upon removal of the SECIS element. See text for further details.

Article Snippet: The RF1-depleted E. coli strain C321.ΔA strain developed by others ( 39 ) and used here for selenoprotein production was obtained from Addgene (catalogue no. 488998).

Techniques: Construct, Recombinant, Purification, Expressing, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depleted Escherichia coli Host Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation *

doi: 10.1074/jbc.M117.776310

Figure Lengend Snippet: Comparison of the method developed here with other approaches for expression of heterologous selenoproteins in E. coli FDH is formate dehydrogenase; EF-Tu is elongation factor Tu.

Article Snippet: The RF1-depleted E. coli strain C321.ΔA strain developed by others ( 39 ) and used here for selenoprotein production was obtained from Addgene (catalogue no. 488998).

Techniques: Expressing, Luciferase, Activity Assay, Mass Spectrometry, Purification, Functional Assay, Produced, In Vivo, Construct

Journal: Frontiers in Microbiology

Article Title: Comparative Analyses of the Transcriptome and Proteome of Escherichia coli C321.△A and Further Improving Its Noncanonical Amino Acids Containing Protein Expression Ability by Integration of T7 RNA Polymerase

doi: 10.3389/fmicb.2021.744284

Figure Lengend Snippet: Bacterial strains and plasmids used in this study.

Article Snippet: E. coli C321.ΔA , E. coli MG1655Δ ( ybhB-bioAB )::[λcI857 N( croea59 ):: tetR-bla ] Δ prfA Δ mutS :: zeoR ; all 321 TAG codons changed to TAA , Addgene (ID: 48998).

Techniques: Plasmid Preparation, CRISPR, Sequencing, Mutagenesis

Journal: Frontiers in Microbiology

Article Title: Comparative Analyses of the Transcriptome and Proteome of Escherichia coli C321.△A and Further Improving Its Noncanonical Amino Acids Containing Protein Expression Ability by Integration of T7 RNA Polymerase

doi: 10.3389/fmicb.2021.744284

Figure Lengend Snippet: The transcriptome profiles of Escherichia coli C321.ΔA and its parent strain. (A) A venn diagram of shared and unique of DEGs between E. coli C321.ΔA and its parent strain. (B) Up- and down-regulated expressed gene count. (C) A scatter plot represents the overall changes of the transcriptomic profiles of E. coli C321.ΔA. The horizontal and vertical coordinates represent the expression of genes in the two strains. The values of the horizontal and vertical coordinates are logarithmic, with each point representing a specific gene. The red points in the graph indicate significantly up-regulated genes, the green points indicate significantly down-regulated genes, and the gray points are non-significantly different genes. When all genes are mapped, the closer the points are to zero, the lower the expression; those points that deviate more from the diagonal indicate that the gene is more differentially expressed between the two samples. (D) Functional classification of the DEGs according to COG. In the horizontal coordinates, the functional classification names were shown. (E) Gene Ontology (GO) annotations analyses of the DEGs. (F) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs. The vertical axis represents the pathway name, and the horizontal axis represents the Rich factor. The size of the dots indicates the number of genes in the pathway, while the color of the dots corresponds to the different FDR-value ranges.

Article Snippet: E. coli C321.ΔA , E. coli MG1655Δ ( ybhB-bioAB )::[λcI857 N( croea59 ):: tetR-bla ] Δ prfA Δ mutS :: zeoR ; all 321 TAG codons changed to TAA , Addgene (ID: 48998).

Techniques: Expressing, Functional Assay

Journal: Frontiers in Microbiology

Article Title: Comparative Analyses of the Transcriptome and Proteome of Escherichia coli C321.△A and Further Improving Its Noncanonical Amino Acids Containing Protein Expression Ability by Integration of T7 RNA Polymerase

doi: 10.3389/fmicb.2021.744284

Figure Lengend Snippet: The proteome profiles of E. coli C321.ΔA and its parent strain. (A) SDS-PAGE analysis of the whole cell proteins of the strain E. coli C321.ΔA and its parent strain. (B) Differential volcano plot of different expressed proteins. The horizontal coordinate is the value of the fold change in protein expression between the two strains, and the vertical coordinate is the statistical test value of the difference in gene expression. Each point in the graph represents a specific protein, the further to the left and up the more significant the difference in expression. (C) Prediction analysis of subcellular localization of the significantly different expressed proteins. (D,E) GO enrichment analyses of the different expressed proteins. (F,G) KEGG pathways analysis of the different expressed proteins.

Article Snippet: E. coli C321.ΔA , E. coli MG1655Δ ( ybhB-bioAB )::[λcI857 N( croea59 ):: tetR-bla ] Δ prfA Δ mutS :: zeoR ; all 321 TAG codons changed to TAA , Addgene (ID: 48998).

Techniques: SDS Page, Expressing, Gene Expression

Journal: Frontiers in Microbiology

Article Title: Comparative Analyses of the Transcriptome and Proteome of Escherichia coli C321.△A and Further Improving Its Noncanonical Amino Acids Containing Protein Expression Ability by Integration of T7 RNA Polymerase

doi: 10.3389/fmicb.2021.744284

Figure Lengend Snippet: Integration of T7 RNA polymerase gene in E. coli C321.ΔA. exp and verification. (A) Display of the overall strategy used in this study to integrate T7 RNA polymerase into the genome of E. coli C321.ΔA.exp. The crossing lines mean that the LacZ gene in the strain C321. exp is replaced by the T7 RNAase gene amplified from the BL21 genome. (B) PCR verification of the successful integration of T7 RNA polymerase in the genome of E. coli C321.ΔA.exp. (C) Growth curves of the E. coli strains used for recombinant protein expression in this study. (D) Expression of GFP in E. coli C321.ΔA.exp and E. coli C321.ΔA.exp T7 with plasmids pBAD24- GFP and pET26b- GFP .

Article Snippet: E. coli C321.ΔA , E. coli MG1655Δ ( ybhB-bioAB )::[λcI857 N( croea59 ):: tetR-bla ] Δ prfA Δ mutS :: zeoR ; all 321 TAG codons changed to TAA , Addgene (ID: 48998).

Techniques: Amplification, Recombinant, Expressing

Journal: Life

Article Title: A Programmable Finite-Replicated Organism Framework for Balanced Safety and Functionality

doi: 10.3390/life15091381

Figure Lengend Snippet: Escape frequency analysis of each suicide module and finite replication dynamics of SerS.F213 FROs. ( a ) Suicide module design schematic featuring dual TAG insertions in essential genes. With the gene editing strategy, the E. coli essential genes ( dnaA , murG , and serS ) were inserted with two TAGs, forming the suicide module (Suicide Module A). ( b ) Quantitative escape frequency measurement expressed as CFU ratio ±Cl2Y supplementation (Mean ± SD, N = 7 biological replicates). Escape frequency was quantified as the ratio of escape mutant colony-forming units (CFUs) to total viable CFUs. ( c ) Engineering strategy for SerS.F213 FROs through rescue module insertion. Two plasmids expressing the ncAA orthogonal translation system and storage protein RFP carrying eight TAGs were constructed. When the exogenous supply of ncAA is interrupted, the storage protein RFP with TAGs degrades and releases the expression of genes necessary for ncAA supply. ( d ) The finite replication characteristics of SerS.F213 FROs. Left panel: growth generation quantification (top) and generational distribution (bottom). The SerS.F213-engineered FROs demonstrated finite proliferation, sustaining four generations of growth before growth arrest, while rescue-module-deficient controls (NC) showed unrestricted proliferation (mean ± SD; **** p < 0.0001, two-tailed Student t -test). Right panel: Microscopic imaging demonstrates finite replication phenotypes in serS -modified FRO. The bacterial growth was observed and filmed every 0, 1, 3, 6, 9, and 12 h.

Article Snippet: The E. coli C321.ΔA exp strain (Addgene #87359) (Lajoie MJ, 2013) was cultured in Luria–Bertani medium (10 g/L NaCl, 10 g/L tryptone, 5 g/L yeast extract) supplemented with antibiotics: ampicillin (100 μg/mL), kanamycin (50 μg/mL), chloramphenicol (25 μg/mL), and spectinomycin (50 μg/mL).

Techniques: Mutagenesis, Expressing, Construct, Two Tailed Test, Imaging, Modification

Journal: Synthetic and Systems Biotechnology

Article Title: A linear DNA template-based framework for site-specific unnatural amino acid incorporation

doi: 10.1016/j.synbio.2021.07.003

Figure Lengend Snippet: Screening of 14 cell extracts. In this experiment, pPaF was selected for the UNAA embedding screening of 14 kinds of cell extracts. A total of 14 extracts were selected, including K-12 series (K-12, K-12 ΔtnaA, K-12 ΔtnaAΔsdaB), commercial series (BL21 (DE3), BL21 ΔserB, Rosetta (DE3), Rosetta-gami B (DE3), Origami, Origami B), rEc series (rEc. 13, rEc. 13. ΔA), EcAR7 series (EcAR7 ΔA ΔSer), C321 series (C321, C321. ΔA).

Article Snippet: ΔA [ ] (Addgene #69495)), EcAR7 series (EcAR7 ΔA ΔSer [ ]) (Addgene #52055), C321 series (C321 [ ] (Addgene #48999), C321.

Techniques:

Journal: Synthetic and Systems Biotechnology

Article Title: A linear DNA template-based framework for site-specific unnatural amino acid incorporation

doi: 10.1016/j.synbio.2021.07.003

Figure Lengend Snippet: Screening of 14 cell extracts. In this experiment, pPaF was selected for the UNAA embedding screening of 14 kinds of cell extracts. A total of 14 extracts were selected, including K-12 series (K-12, K-12 ΔtnaA, K-12 ΔtnaAΔsdaB), commercial series (BL21 (DE3), BL21 ΔserB, Rosetta (DE3), Rosetta-gami B (DE3), Origami, Origami B), rEc series (rEc. 13, rEc. 13. ΔA), EcAR7 series (EcAR7 ΔA ΔSer), C321 series (C321, C321. ΔA).

Article Snippet: ΔA [ ] (Addgene #69495)), EcAR7 series (EcAR7 ΔA ΔSer [ ]) (Addgene #52055), C321 series (C321 [ ] (Addgene #48999), C321.

Techniques: