Journal:

Article Title: DapE Can Function as an Aspartyl Peptidase in the Presence of Mn 2+

doi: 10.1128/JB.185.16.4748-4754.2003

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

Article Snippet: Liquid cultures were aerated by shaking on a rotary shaker, and all growth incubations were at 37°C. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Genotype or description Source or reference Strains S. enterica serovar Typhimurium TN1379 leuBCD485 Lab collection TN1715 leuBCD485 supQ302 Δ( proAB pepD ) pepN90 pepA16 pepB11 pepP1 pepQ1 pepT1 Lab collection TN5307 As TN1379 plus vector pSE380 Lab collection TN5538 leuBCD485 iaaA1 :: chl Lab collection TN5771 As TN1379 plus pKD46 This study TN5834 leuBCD485 pepT7 :: MudJ iadA50 :: chl This study TN5860 As TN1715 plus iadA50 :: chl This study TN5862 As TN5860 plus iadA100 This study TN5874 leuBCD485 pepE25 :: chl This study TN5875 As TN5862 plus pepE25 :: chl This study TN5879 As TN5875 plus pepE50 This study TN5889 As TN5879 plus iaaA1 :: chl This study TN5891 As TN5889 plus Δ sitA This study TN5893 leuBCD485 pepT7 :: MudJ plus pDK46 This study TN5896 As TN5891 plus dapE2 This study TN5909 As TN5891 plus dapE1 :: kan This study TN5910 As TN1379 plus dapE1 :: kan This study TN5911 As TN5889 plus dapE1 :: kan This study TN5934 As TN5911 plus pSE380 This study TN5935 As TN5911 plus pCM655 This study TN5936 As TN5918 plus pSE380 This study E. coli DH5α φ80 lacZ ΔM15 Δ( lacZYA ) U169 endA1 hsdR17 (r k − m k − ) supE44 thi-1 recA1 gyrA96 relA1 Gibco, BRL Top10F′ F − mcr A Δ( mrr-hsdRMS-mcrBC ) φ80 lacZ ΔM15 Δ lacX74 rec A1 deoR araD139 d(ara-leu)7697 galU galK rpsL (Str r ) endA1 nupG Invitrogen BL21 F − ompT hsdSb (r B − m B − ) gal dcm (DE3) Novagen TN5697 BL21/pCM652 This study Plasmids pCR 2.1-TOPO Cloning vector for PCR products Invitrogen pET28 T7 promoter Novagen pSE380 tac promoter Stratagene pKD46 λ Red recombinase B. L. Wanner pKD2 kan flanked by FRT recombinase sites B. L. Wanner pKD3 cat flanked by FRT recombinase sites B. L. Wanner pCM651 dapE-his 6 on pCR2.1 TOPO This study pCM652 dapE-his 6 on pET28 This study pCM653 dapE on pCR2.1 TOPO This study pCM655 dapE on pSE380 This study Open in a separate window Bacterial strains and plasmids used in this study

Techniques: Plasmid Preparation, Clone Assay

Journal: bioRxiv

Article Title: Tandem histone-binding domains enhance the activity of a synthetic chromatin effector

doi: 10.1101/145730

Figure Lengend Snippet: ELISA was used to determine binding affinity and specificity of Pc 2 TF variants that were expressed in a bacterial cell-free expression system. (A) Map of the expression vector and open reading frames (ORFs). Fusion-encoding ORFs were cloned in the pET28 vector at BamHI and XhoI. (B) Real-time detection of mCherry fluorescence was used to determine expression of recombinant protein in TXTL in a 96-well PCR plate in a Roche thermal cycler. n = 1 for Pc Δ TF and TXTL without DNA, n = 3 for others, shaded regions = SDM. (C) The bar chart shows binding of TXTL-expressed fusion proteins (or plasmid-free blank TXTL) with trimethyl-K27, unmodified, or off-target (acetyl-K27) histone H3 peptides. Values for each TXTL sample are normalized to unmodified H3 (n = 3, bars = SDM).

Article Snippet: BamHI/XhoI-digested inserts and 50 - 75 ng BamHI/XhoI linearized pET28(+) vector were ligated at a 3:1 molar ratio in a 20 μL reaction as described in the New England Biolabs (NEB) protocol for T4 ligase (M0202).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Expressing, Plasmid Preparation, Clone Assay, Fluorescence, Recombinant

Journal: bioRxiv

Article Title: Tandem histone-binding domains enhance the activity of a synthetic chromatin effector

doi: 10.1101/145730

Figure Lengend Snippet: A bivalent PCD fusion peptide shows enhanced H3K27me3 binding in vitro. (A) For high-yield expression, E. coli was transformed with pET28 plasmids encoding Pc Δ TF (negative control), PcTF (single PCD), and the Pc 2 TF containing the flexible linker (GGGGS) 4 . Native polyacrylamide gel electrophoresis (PAGE) of over-expressed proteins purified from E. coli. (C) ELISA was used to detect interaction of purified proteins with histone peptides that were trimethylated, unmodified, or acetylated at lysine 27. The bar chart shows mean HRP signal (n = 4 replicate ELISAs, bars = SDM).

Article Snippet: BamHI/XhoI-digested inserts and 50 - 75 ng BamHI/XhoI linearized pET28(+) vector were ligated at a 3:1 molar ratio in a 20 μL reaction as described in the New England Biolabs (NEB) protocol for T4 ligase (M0202).

Techniques: Binding Assay, In Vitro, Expressing, Transformation Assay, Negative Control, Polyacrylamide Gel Electrophoresis, Purification, Enzyme-linked Immunosorbent Assay