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Ptyb21

Manufactured by New England Biolabs
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PTYB21 is a lab equipment product manufactured by New England Biolabs. It is a plasmid used for the expression and purification of recombinant proteins in E. coli.

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Lab products found in correlation

Bamhi, by New England Biolabs (1 mentions) Geneart, by Thermo Fisher Scientific (1 mentions) Nanodrop one microvolume uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions) Pbr322, by New England Biolabs (1 mentions) E coli jm109, by Promega (1 mentions) Puc19, by New England Biolabs (1 mentions) Zymopure plasmid maxiprep kit, by Zymo Research (1 mentions)

6 protocols using ptyb21

1

Recombinant expression of ToxR and ToxS

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DNA corresponding to the amino acid sequences of V. cholerae El Tor N16961 ToxR (VC0984) T199-E294 and ToxS (VC0983) S25-S173 were ordered from DNA2.0 and inserted into pTYB21 (NEB). The constructs were digested out of the DNA 2.0 plasmids using NdeI and BamHI (NEB) and inserted into pTYB21 in frame with the N-terminal chitin binding domain-intein tag. The plasmid sequences were verified by sequencing. This resulted in constructs that contained an additional five amino acids on the N-terminus when cleaved from the intein tag. The domains were expressed using Codon plus (DE3) pRIL cells (Agilent).
Midgett C.R., Almagro-Moreno S., Pellegrini M., Taylor R.K., Skorupski K, & Kull F.J. (2017). Bile salts and alkaline pH reciprocally modulate the interaction between the periplasmic domains of Vibrio cholerae ToxR and ToxS. Molecular microbiology, 105(2), 258-272.
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2

Plasmid pTYB21 Characterization

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The circular plasmid pTYB21 (7514 bp) in 10 mM Tris and 1 mM EDTA was purchased from New England Biolabs and used for translocation and AFM investigations without further purification.
Krueger E., Shim J., Fathizadeh A., Chang A.N., Subei B., Yocham K.M., Davis P.H., Graugnard E., Khalili-Araghi F., Bashir R., Estrada D, & Fologea D. (2016). Modelling and Analysis of Intercalant Effects on Circular DNA Conformation. ACS nano, 10(9), 8910-8917.
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3

Recombinant Expression of Bacterial Virulence Factors

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The gene fragments transcribing the mature form of the wild-type LmPC-PLC (UniProt ID P33378), LLO (UniProt ID P13128) and CpPC-PLC (UniProt ID Q0TV31) were prepared by using genomic DNA isolates kindly provided by the Faculty of Veterinary Medicine at the University of Ljubljana, Slovenia. The gene encoding the mature wild-type BcPC-PLC (UniProt ID P09598) and proLmPC-PLC (UniProt ID P33378) were purchased from Invitrogen (USA). Individual PC-PLC gene fragments were inserted into plasmid pTYB21 (NEB, USA), downstream and in-frame of the intein tag with chitin binding domain. LLO gene was inserted in pPROEX HTb vector downstream of the His6-tag and TEV recognition site. All LmPC-PLC mutants were prepared using site directed mutagenesis by PCR69 (link). All constructs were verified by nucleotide sequencing (Eurofins Genomics, Luxembourg). List of oligonucleotides used in cloning and PCR mutagenesis is displayed in Supplementary Table 3.
Petrišič N., Adamek M., Kežar A., Hočevar S.B., Žagar E., Anderluh G, & Podobnik M. (2023). Structural basis for the unique molecular properties of broad-range phospholipase C from Listeria monocytogenes. Nature Communications, 14, 6474.
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4

Synthetic Biosynthetic Gene Constructs

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Full-length P. somniferum PsTyDC1 native coding sequence was synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). Codon optimization of PsONCS3 and TfNCS nucleotide sequences60 (link) for expression in E. coli was assisted by Codon Optimization OnLine (COOL)61 (link), resulting in the coding sequences shown in Supplementary Table 9, and the selected sequences were synthesized by IDT. The native sequence of full-length P. somniferum NMCH isoform 1 (PsNMCH-I1) was also synthesized by IDT.
Native coding sequences of full-length PsPDC1, full-length Ps2HCLL, and N-terminal truncated PsPDC2 were synthesized and cloned into pBAD-DEST49 (LifeSensors Inc., Malvern, PA, USA) via the Gateway cloning system by GeneArt (Invitrogen, Waltham, Massachusetts, USA). Native coding sequences of full-length EcNMCH, AtATR2, and P. somniferum CPR-like (PsCPR-L) were synthesized and subcloned into the pMA vector by GeneArt (Invitrogen). Native coding sequences of full-length PsTyDC6 and N-terminal truncated PsPDC1-IX1 were synthesized and cloned into pTYB21 (NEB) by GenScript (Piscataway, NJ, USA).
Vavricka C.J., Takahashi S., Watanabe N., Takenaka M., Matsuda M., Yoshida T., Suzuki R., Kiyota H., Li J., Minami H., Ishii J., Tsuge K., Araki M., Kondo A, & Hasunuma T. (2022). Machine learning discovery of missing links that mediate alternative branches to plant alkaloids. Nature Communications, 13, 1405.
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5

Cloning and Purification of AdcA-NTD

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The coding sequence of the extracellular domain of adcA without the secretion signal sequence (amino acids 32-320) from strain MGAS10870 was amplified and cloned into NdeI and BamHI restriction sites of E. coli overexpression vector pTYB21 (New England BioLabs). The details of AdcA-NTD purification is described in the Supplemental Experimental Procedures.
Makthal N., Nguyen K., Do H., Gavagan M., Chandrangsu P., Helmann J.D., Olsen R.J, & Kumaraswami M. (2017). A Critical Role of Zinc Importer AdcABC in Group A Streptococcus-Host Interactions During Infection and Its Implications for Vaccine Development. EBioMedicine, 21, 131-141.
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6

Plasmid Isolation and Characterization

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Original solutions of ampicillin-resistant plasmids pUC19 (2686 bp), pBR322 (4361 bp), and pTYB21 (7514 bp) were purchased from New England BioLabs (Ipswich, MA). JM109 E. coli (Promega, Madison, WI) were transformed with the plasmids and grown in Luria-Bertani (LB) broth dosed with 100 μg/mL ampicillin. Plasmids were extracted with ZymoPure Plasmid Maxiprep Kit (Zymo, Irvine, CA) by following manufacturer's instructions, and suspended in UltraPure DNase/RNase-Free Distilled Water (DIW). Concentrations and purity of plasmid stock solutions were determined with a NanoDrop One Microvolume UV–Vis Spectrophotometer (Thermo Scientific, Waltham, MA) and DNA integrity was assessed by gel electrophoresis.
Cochran J.P., Zhang L., Parrott B.B, & Seaman J.C. (2024). Plasmid size determines adsorption to clay and breakthrough in a saturated sand column. Heliyon, 10(9), e29679.
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