Tag: BL21

Abstract The mitochondrial genome, mtDNA, is present in multiple copies in cells and encodes essential subunits of oxidative phosphorylation complexes. mtDNA levels have to change in response to metabolic demands and copy number alterations are implicated in various diseases. The mitochondrial HMG-box proteins Abf2 in yeast and TFAM in mammals…

Deep Mutational Scanning profiles beneficial substitutions in the RBP tip domain (A-B) Heatmaps showing normalized functional scores (FN) of all substitutions (red gradient) and wildtype amino acids (FN=1 and black dot upper left) at every position after incubation with E. coli BL21 in (A) microgravity and (B) after terrestrial incubation….

Abstract While the majority of circRNAs are formed from infrequent back-splicing of exons from protein coding genes, some can be produced at quite high level and in a regulated manner. We describe the regulation, biogenesis and function of circDOCK1(2–27), a large, abundant circular RNA that is highly regulated during epithelial-mesenchymal…

Ethics statement All mice procedures in this study were performed under an animal protocol approved by the Institutional Animal Care and Use Committee guidelines of Westlake University. The procedures and protocols for glioma patients were approved by the institutional review board of Beijing Tiantan Hospital. Informed consent was obtained from…

Statistics and reproducibility Anti-AMP IP for LC-MS/MS analysis was performed in three independent biological replicates (n = 3). TSA assay data represents technical triplicates. Time-resolved (de)AMPylation analyzed by LC-MS was performed as biological triplicates. CD measurements were performed in technical triplicates. Anisotropy data is shown as technical triplicates. Anisotropy measurements were repeated…

     Preparation and in vitro Activity of Anti-EpCAM Immunotoxin LIU Yu-ping1,DENG Chang-ping2,MA Xing-yuan2,LIU Qiu-li1,BAO Wen1,ZHENG Wen-yun1,**() 1 Shanghai Key Laboratory…

Bacterial strains and phages Escherichia coli DH5a was routinely grown in Lysogeny broth (LB) medium and used for plasmid cloning, while E. coli BL21 (DE3) was used for protein production and the in vivo assays. E. coli phages T5, T7 and Lambda-vir are gifts from Shi Chen lab (Wuhan University,…

Identification of Cutinolytic Esterase from Microplastic-Associated Microbiota Using Functional Metagenomics and Its Plastic Degrading Potential Atıf İçin Kopyala Adıgüzel A. O., Şen F., Könen-Adıgüzel S., KIDEYŞ A. E., KARAHAN A., Doruk T., …Daha Fazla Molecular Biotechnology, 2023 (SCI-Expanded) …

Cloning Supplementary Table 1 shows the synthetic gene, DNA and RNA oligonucleotide sequences used in this study. The synthetic genes encoding B.fragilis Cas6, CorA, NrN and C. botulinum SAM lyase purchased as g-blocks (IDT) were codon-optimized for expression in E. coli C43 (DE3) via the vector pEhisV5TEV, which encodes eight…

Parasites and animals PSCs were collected from hydatid cysts isolated from the liver of sheep infected with E. granulosus s.l. from a slaughterhouse in Sichuan province, China. The PSCs and cyst walls were separated and treated as described in previous reports [22]. The viability and molecular genotyping of PSCs were…

Plant material and constructs A. thaliana lines of Columbia-0 (Col-0) ecotype were employed, including WT, toc159 (refs. 58,59) and cct8-2 (ref. 60). Seeds underwent surface sterilization and germination on solid 0.5× Murashige and Skoog (M&S) medium with vitamins, lacking sucrose. Plants were incubated in a growth chamber at 22 °C under…

Introduction The primary cilium (9 + 0 architecture) is a microtubule-based immotile sensory organelle in vertebrates (Tucker et al, 1979). Enormous progress has been made in the past several decades concerning our understanding of the structure and functions of the primary cilium (Sung & Leroux, 2013). The subdomain between the…

Cultivations and harvest Cupriavidus necator strain DSMZ 428 was grown in Ralstonia Minimal Media (RMM) with 100 mM HEPES pH 7.5 under chemostat conditions in a Photon Systems Instruments Multi-Cultivator MC-1000 OD. Each reactor tube was set up to a volume of 55 mL, OD600 0.05 and 3.5 g/L fructose. Once growth ceased,…

MenAT1 sequence analysis Analysis of gene neighbourhoods for rv0078B (menA1) was performed using default settings in FlaGs (www.webflags.se/). Output sequences for MenA1 and cognate MenT1 homologues were then used to perform sequence alignments using MUSCLE (www.ebi.ac.uk/Tools/msa/muscle/), then formatted in Jalview (www.jalview.org/), sorting by pairwise alignment. Residues of interest were then…

replying to G. Benn et al. Nature Communications doi.org/10.1038/s41467-023-40275-0 (2023) Benn et al. comment that the hexagonal lattices observed in outer membrane (OM) patches upon the addition of polymyxin might be formed by outer membrane proteins (OMPs). They assume that lipopolysaccharides (LPS) protrude above the OMPs, which might hide the…

Mice and parasites The strains RH, Pru, wh3, and wh6, utilized in the present investigation, were generously provided by the Anhui Key Laboratory of Zoonoses, Anhui Medical University. The DNA of C. parvum and Babesia were procured from Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Furthermore, Plasmodium was…

Cell culture CHO cells (a kind gift provided by Prof. Tom K. Hei, Columbia University, USA) were cultured in DMEM/F12 medium (Gibco, USA) containing 10% fetal bovine serum (FBS, Bio-west, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Beyotime, China), at 37 °C in a 5% CO2 incubator. Vector construction To capture…

Constructs and expression of human SERINC proteins SERINC3 The gene that encodes human SERINC3 (Genscript-OHu02717D) was inserted upstream of a thrombin protease cleavable linker (LVPRGS) and Strep II epitope (WSHPQFEK) in a modified pFastBac vector by In-Fusion cloning (Clontech). Mutagenesis using the QuikChange Site-Directed Mutagenesis kit (Agilent) was performed to…

To predict the structure and antigenic epitope of the serine protease inhibitor 1 (Ss-SRPN-1) protein using bioinformatics tools, and to construct prokaryotic expression plasmids for expression of recombinant Ss-SRPN-1 protein, so as to provide the basis for unraveling the function of the Ss-SRPN-1 protein.The amino acid sequence of the Ss-SRPN-1…

Bacterial strains and culture conditions Escherichia coli strain DH5α (Thermo Fisher) was used for the propagation of plasmids, whereas E. coli BL21 DE3 (Novagen) was used for the expression and purification of ResR/McdR protein. Mtb Erdman was obtained from Dr. Ramandeep Singh at THSTI, India, and Mtb H37Rv mc2 790242…

Introduction Helicobacter pylori became a concern in 1979 when John Warren, a pathologist in Perth, Western Australia, discovered that the inflamed gastric mucosa was covered with bacteria that looked like Campylobacter.1 Warren and his colleague Barry Marshall cultured the first batch of these bacteria, newly named Helicobacter pylori, subsequently from…

Cell lines and culture A549-hACE2 [a gift from Dr. Ben tenOever3], 293 T, HEK293, A549, and 3T3-L1 pre-adipocyte cell lines (refer to Supplementary Table 1) were cultured in DMEM and 10% FBS. U937, Calu-3, and HCT116 cells were cultured in RPMI, EMEM, and McCoy’s 5 A media, respectively, with 10% FBS. Caco-2 cells…

Expression and purification of human PTH1R The plasmid encoding human PTH1R (GenBank identifier: U17418.1; residues 27–491) was constructed and purified as previously reported17. The construct was expressed in HEK293 GnTI (N-acetylglucosaminyltransferase I-negative) cells (American Type Culture Collection, CRL-3022) using the BacMam system (Thermo Fisher Scientific), and the cells were grown…

Abstract De novo protein design enhances our understanding of the principles that govern protein folding and interactions, and has the potential to revolutionize biotechnology through the engineering of novel protein functionalities. Despite recent progress in computational design strategies, de novo design of protein structures remains challenging, given the vast size…

Protein expression, mutagenesis and purification Xenopus laevis histones for nucleosome assembly were overexpressed in the Escherichia coli BL21(DE3) pLysS strain and purified from inclusion body as previously described48. The cells were grown in LB medium at 37 °C and induced with 1 mM IPTG when OD600 reached 0.6. After 3 h of expression,…

 NZYEasy Cloning & Expression kit X was designed to allow directional cloning of any PCR-generated fragment or synthetic gene into the linearized pHTP10 Escherichia coli expression vector (contains the N-utilization substance, NusA, as fusion tag). Cloning proceeds in a single ligase-independent reaction mediated by the NZYEasy enzyme mix. Vector-complementary overhangs…

Constructs MOTH domains of human TALI (23-123), Otoraplin (18-128), and MIA (19-131), and TANGO1 (30-139) from Drosophila melanogaster (dmTANGO1(30-139)) were expressed from codon-optimized sequences in a modified pQE40 expression vector36 in M15 pRep4 E. coli strain. Human TANGO1 (21-131) (hsTANGO1 (21-131)) and TANGO1 (21-151) (hsTANGO1 (21-151)) were expressed from codon-optimized…

Cell culture and reagents HEK293 and HeLa cells (American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 2 mM GlutaMAX‐1, 100 μg/mL streptomycin and 100 U/mL penicillin (complete growth medium) at 37 °C under 5% CO2. HeLa cells stably expressing GFP-TIFA were previously described8. Cells…

Plasmids The mingag fusion protein was generated by fusing a codon-optimised HIV-1 subtype B gp41 miniprotein (“min”) containing a fragment of the HR2 domain, the membrane proximal external region (MPER) and the transmembrane domain (TM) (HXB2: 8057-8345 bp)36 with a codon-optimised HIV-1 subtype B gagHXB2. A flexible GGGS linker was inserted…

Purification of T7Select 10-3b phages The T7Select 10-3b phage vector (Novagen, Merck) was used in this study. Non-recombinant/recombinant T7Select 10-3b phages were purified from the lysate of infected E. coli strain BL5403 by PEG-8000 precipitation as per the method described in T7 select system manual. The pellet was suspended in…

Plasmid construction The PVCpnf structural and accessory region (pvc1-16) and payload and regulatory region (Pdp1, Pnf and regulatory genes PAU_RS16570-RS24015) were synthesized de novo (GenScript) and cloned into pAWP78 and pBR322 backbones, respectively. All manipulations involving payload and regulatory plasmids (pPayload) involved standard PCR amplification with Phusion Flash 2x Master…

SnapGene in silico control digestion Molecular Techniques Lab AUC/DuBa June 2021 In silico digestion using SnapGene During the previous assignment in SnapGene you have used PCR and Golden Gate Assembly to recreate your constructs in silico. By now you have also performed the PCR in vitro, while the Golden Gate…

Recombinant peptide production Recombinant expression of Xt3a, Xt3a-D1, Xt3a-D2 and IpTxA was performed using an E. coli expression system. A gene encoding the peptide was subcloned into an expression vector containing a coding region with poly-histidine purification tag as well as a solubility tag (MBP for Xt3a and SUMO for…

Here we provide a tool that directly selects biallelically edited knock-ins using cell-surface displays, thereby eliminating the need for extensive clonal verification. By doing so SNEAK PEEC greatly reduces the considerable effort currently expended to obtain biallelically edited clones. The ability to generate a clonal population with multiple edits is…

Sequence analysis of S2-ALDH BLAST screening of the Flavobacterium PL002 genome sequence42 led to identification of a coding region homologous to aldehyde dehydrogenase gene (1365 bp) coding for the hypothetical enzyme S2-ALDH of 454 amino acids, with calculated MW 49,062 (Supplementary Fig. 1S). Phylogenetic analysis of the S2-ALDH amino acid sequence in…

Cell culture Human glioblastoma cells (U87MG and U87MG-Luc) and human embryonic kidney cells (HEK293) were obtained from the American Type Culture Collection (Manassas, Va.). Cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (Gibco™ Fetal Bovine Serum South America, Thermo Scientific Fisher-US), 2 mM l-glutamine, 50 U/ml…

DNM filtering in 100,000 Genomes Project We analysed DNMs called in 13,949 parent–offspring trios from 12,609 families from the rare disease programme of the 100,000 Genomes Project. The rare disease cohort includes individuals with a wide array of diseases, including neurodevelopmental disorders, cardiovascular disorders, renal and urinary tract disorders, ophthalmological…

Introduction Antibody-drug conjugates (ADCs) have been studied for decades as a promising cancer treatment. As compared to conventional chemotherapy, ADCs have a larger therapeutic window because of their ability to target antigen-expressing tumor cells.1 As ADC drug development technology continues to evolve, several ADC drugs are being introduced to the…

Keyword fragment (multi-family coverage) (14) Protein construct that is shorter than the full-length protein but corresponds to two or more protein family domains. Nodes  PR domain  (8000876)  2FY8 A:115-336 O27564 115-336 Calcium-gated potassium channel mthK Methanothermobacter thermautotrophicus (145262) Derived from SCOP domain 134345  PR domain  (8002280)  1OFC X:697-978 Q24368 697-978Chromatin-remodeling complex ATPase chain Iswicomplexed with g4d, glc, gol Drosophila melanogaster…

Significance Interferon (IFN)-mediated antiviral responses serve as the first line of the host innate immune defense against viral infection. Here we identify a previously uncharacterized protein designated phospholipase A2 inhibitor and LY6/PLAUR domain-containing protein (PINLYP), which is essential for embryonic development and plays an important role in type I IFN…

Computational design Protein modeling and design was performed with Rosetta version 3.5 (2015.19.57819)35,37. Python and shell scripts for generating input from Rosetta and analyzing from Rosetta are available at: github.com/raman-lab/biosensor_design The high-resolution TtgR structure co-crystalized with tetracycline was selected as the starting point for computational design (PDB: 2UXH)28. The structure…

Introduction Carrión’s disease is a vector-borne biphasic illness restricted to the South American Andes, with Peru as the most affected country. 1 Gomes C Pons MJ Del Valle Mendoza J Ruiz J Carrion’s disease: an eradicable illness?. The causative agent, Bartonella bacilliformis, is transmitted to humans by sandflies (Lutzomyia spp)….

INTRODUCTION Charles R. Darwin documented inbreeding depression as growth disadvantages from self-fertilization compared to outcrossing in many plants (1). Prevailing hypotheses suggest that inbreeding depression results from the exposure of deleterious recessive alleles and/or loss of overdominant alleles due to increased homozygosity (2, 3) or reduced recombination frequency in some…