Plasmid DNA is a versatile and indispensable tool in modern biology, enabling advancements in genetics, medicine, biotechnology, and beyond. Its applications continue to expand as new technologies and methodologies emerge.

What are the Quality Requirements for Plasmid DNA in Different Applications?

Plasmid DNA is widely used in various applications, each with distinct purity and quality requirements. The supercoiled DNA ratio and endotoxin levels vary depending on whether the plasmid is used for research, cell transfection, or clinical trials.

Research-Grade Plasmid DNA

Application: General molecular biology research, cloning, PCR, in vitro transcription, bacterial transformation.

• Purity: A260/A280 ratio of 1.8–2.0 for protein contamination control.

• Supercoil Ratio: Not strictly required, but a moderate percentage (>70%) is preferred for stable expression.

• Endotoxin Level: Endotoxin concentrations below0.1-10 EU/μg DNA are usually required, depending on the type of experiment.

Transfection-Grade Plasmid DNA

Application: Transient or stable transfection of mammalian cells for protein expression, CRISPR genome editing, or viral packaging.

• Purity: High purity with A260/A280 ratio of 1.8–2.0 and A260/A230 > 2.0 to ensure minimal RNA, protein, and salt contamination.

• Supercoil Ratio:≥80% supercoiled plasmid is preferred, as higher supercoiling enhances transfection efficiency and stability in cells.

• Endotoxin Level:Endotoxin levels are typically required to be <0.1 EU/µg, with certain highly sensitive applications demanding even lower levels,≤0.005 EU/µg. High endotoxin concentrations can trigger inflammatory responses and reduce transfection efficiency.

GMP-Grade / Clinical-Grade Plasmid DNA

Application: Used in gene therapy, vaccine production, CAR-T cell therapy, and clinical trials. Requires compliance with Good Manufacturing Practices (GMP).

• Purity: Ultra-pure plasmid with strict A260/A280 ratio of 1.8–2.0 and A260/A230 > 2.0, ensuring no residual contaminants.

• Supercoil Ratio:≥90%, as high supercoiling improves stability and functionality for therapeutic applications.

• Endotoxin Level: <0.1 EU/µg or even <0.01 EU/µg, as extremely low endotoxin levels are required for clinical safety.

Case Study - Research Grade Plasmid DNA Application

For scientists, accurate and reliable plasmid DNA technical support partners are one of the keys to achieving breakthrough results. We are honored to share with you the latest research results powered by Synbio Technologies.

Title:Facilitation of Mucosal Healing by Estrogen Receptor β in Ulcerative Colitis Through Suppression of Branched-Chain Amino Acid Transport and Subsequent Triggering of Autophagy in Colonic Epithelial Cells

Journal: Advanced Pharmaceutical Sciences and Biotechnology

Impact Factor: 4.4

Collaborating Institution: China Pharmaceutical University

Service Provided by Synbio Technologies: Research Grade Plasmid DNA Preparation

https://doi.org/10.1016/j.apsb.2024.11.014

Abstract: This research is the first to reveal a positive correlation between the activation of estrogen receptor β (ERβ) in colonic epithelial cells and mucosal healing in ulcerative colitis (UC) patients. The findings demonstrate that ERβ activation promotes autophagy in colonic epithelial cells, inhibits branched-chain amino acid (BCAA) transport, and subsequently activates focal adhesion kinase (FAK), accelerating epithelial cell migration and mucosal repair.

Moreover, a UC model in ERβ−/− mice exhibited significantly impaired healing compared to wild-type mice, further confirming the critical role of ERβ in intestinal repair. The study also found that the combination of an ERβ agonist with 5-aminosalicylic acid (5-ASA), a common UC treatment, enhanced its anti-colitis effects, providing a novel therapeutic strategy for ulcerative colitis treatment.

What Value Do the Plasmid DNA we Offer Bring to Research?

We provided the research team with paxillin-pmCherry and pmCherry-EGFP-Light Chain 3 (LC3) plasmids, enabling real-time monitoring and analysis of focal adhesion turnover in live cells.

In the Focal Adhesion Turnover Assay, fluorescently labeled plasmids were used to transfect cells, and real-time tracking and imaging of adhesion sites were performed using an LMS800 confocal fluorescence microscope. This experiment focused on observing how focal adhesions (FAs) assemble and disassemble during cell migration. The application of these plasmids allowed researchers to visually track dynamic changes in focal adhesions, providing crucial insights into the role of ERβ in colonic mucosal repair.

Title: Bergenin, the main active ingredient of Bergenia purpurascens, attenuates Th17 cell differentiation by downregulating fatty acid synthesis

Journal: FASEB Journal

Impact Factor: 14.7

Collaborating Institution: China Pharmaceutical University

Service Provided by Synbio Technologies: Research Grade Plasmid DNA Preparation

https://doi.org/10.1016/j.apsb.2024.11.014

Abstract: Bergenin, the primary active compound of the medicinal plant Bergenia purpurascens, has long been used to treat various Th17 cell-related diseases, such as allergic airway inflammation and colitis. This study explores the potential mechanism by which Bergenin suppresses the Th17 cell response from a cellular metabolism perspective.

In in vitro experiments, Bergenin treatment significantly reduced the differentiation of murine naïve CD4⁺ T cells into Th17 cells. Mechanistically, Bergenin restricts the acetylation of histone H3K27 at the SREBP1 promoter region, leading to the downregulation of SREBP1, a key lipid metabolism regulator. This suppression of fatty acid synthesis subsequently hinders Th17 cell differentiation.

Furthermore, the study revealed that Bergenin directly interacts with and activates SIRT1, a crucial factor in inhibiting fatty acid synthase (FAS) and Th17 cell differentiation. Finally, in a dextran sulfate sodium (DSS)-induced colitis mouse model, Bergenin demonstrated therapeutic potential by effectively suppressing Th17 cell responses via a SIRT1-dependent pathway, offering a novel therapeutic strategy for Th17-related diseases.

What Value Do the Plasmid DNA we Offer Bring to Research?

We provided the research team with SREBP1-related plasmids, assisting them in gene function validation and supporting their mechanistic studies and analysis. 

Future Prospects

Through these two case studies, we can see that plasmid DNA is not only valuable for gene function validation and metabolic mechanism research, but also plays a crucial role in live-cell localization and observation through the construction of fluorescent fusion protein plasmids. Moreover, a diverse range of plasmid vectors can support applications in protein expression, stable cell line/engineered strain construction, viral packaging, mRNA synthesis, vaccine development, and cell and gene therapy. 

We believe that these groundbreaking studies not only provide new perspectives on ulcerative colitis treatment but also offer valuable insights for your research. Synbio Technologies remains committed to delivering cutting-edge gene synthesis and synthetic biology services to researchers worldwide, empowering scientific breakthroughs.

Unlock the Power of High-Quality Plasmid DNA with Synbio Technologies

At Synbio Technologies, we are committed to delivering high-purity, customized plasmid DNA preparation services tailored to your research and therapeutic needs. Our rigorous quality control ensures that plasmid DNA is aseptic, free from RNA and genomic contamination, and manufactured without animal-derived materials.

We offer research-grade and transfection-grade plasmids, with low endotoxin levels available upon request (<0.1 EU/µg,<0.01 EU/µg,<0.005 EU/µg). Whether you're conducting cutting-edge research or performing high-efficiency transfections, our plasmids provide unparalleled reliability and performance. 

Reference:

He Y, Xu D, Zhang J, Liu Y, Liao M, Xia Y, Wei Z, Dai Y. Bergenin, the main active ingredient of Bergenia purpurascens, attenuates Th17 cell differentiation by downregulating fatty acid synthesis. FASEB J. 2024 Oct 15;38(19):e70095. doi: 10.1096/fj.202400961R. PMID: 39373984.

Guo Y, Zhu Y, Zhang J, He Y, Zhao M, Lin H, Wei Z, Xia Y, Dai Y. Facilitation of mucosal healing by estrogen receptor β in ulcerative colitis through suppression of branched-chain amino acid transport and subsequent triggering of autophagy in colonic epithelial cells. Acta Pharm Sin B. 2025 Jan;15(1):168-187. doi: 10.1016/j.apsb.2024.11.014. Epub 2024 Nov 26. PMID: 40041890; PMCID: PMC11873653.

Bower, D.M., Prather, K.L. Development of new plasmid DNA vaccine vectors with R1-based replicons. Microb Cell Fact 11, 107 (2012). https://doi.org/10.1186/1475-2859-11-107