GenStable™ Host Strain Platform

Superior at Pertaining Poly(A) Integrity during Plasmid Propagation

pUC57 and GenScript mRNA Applied Vectors (pGS), containing Poly(A) tails of 100A and 120A with a GFP insert, were constructed and subcultured into GenStable™ Poly(A) and NEB Stable strains over eight generations. Sanger sequencing was conducted at each generation to evaluate the integrity of the Poly(A) sequences. The GenStable™ Poly(A) Strain demonstrated significantly superior performance in maintaining Poly(A) sequence integrity during plasmid preparation. For optimal performance use with a GenScript mRNA Applied Vector.

Ability to Refine Poly(A) Sequences

An mRNA template plasmid possessing a Poly(A) tail of 110 adenines was introduced into the Transformation into GenStable™ Poly(A) Strain for growth and replication. Subsequent Sanger Sequencing was employed to validate the poly(A) sequences. The resulting chromatogram from the sequencing revealed that after growth in the Transformation into GenStable™ Poly(A) Strain, the "noise" (baseline) peaks were significantly diminished, highlighting the capacity of the Transformation into GenStable™ Poly(A) Strain to enhance the clarity of plasmids with elongated poly(A) sequences.

Superior at Pertaining ITR Integrity during Plasmid Propagation

pUC57-GFP plasmid containing wildtype AAV2 ITRs was constructed and subcultured into GenStable™ ITR and NEB Stable strains over 10 generations. SmaI digestion and Nanopore Sequencing was conducted on even-numbered generations to evaluate ITR sequence integrity. GenStable™ ITR Strain demonstrated a significantly superior ability to maintain ITR sequence integrity during plasmid preparation. For optimal performance use with GenScript AAV Applied Vector.

Pertaining ITR regions without Sacrificing Plasmid Yield

	OD600	mg/L	mg/L/OD
NEB Stable Strain	82	190	2.3
GenStable ITR™ Strain	52	396	7.6

The pUC57-GFP plasmid containing wild-type AAV2 ITRs was constructed and cultured in both GenStable™ ITR and NEB Stable strains. Plasmid DNA preparation was carried out using 4 mL (miniprep) and 4 L (bioreactor) scales to evaluate the plasmid yield capabilities of the two strains. GenStable™ ITR showed a significant increase in plasmid yield compared to NEB Stable during preparation.

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