BioVector? ZYCY10P3S2T E. coli Minicircle Producer Strain 大腸桿菌微環(huán)生產(chǎn)菌株Datasheet

Product Name: BioVector? ZYCY10P3S2T Competent Cells
Chinese Name: BioVector? ZYCY10P3S2T 大腸桿菌微環(huán)生產(chǎn)菌株
English Name: BioVector? ZYCY10P3S2T E. coli Minicircle Producer Strain
Product Category: Genetically Engineered Strain; Minicircle DNA Production Strain
Strain Type: Escherichia coli, BW27783-derived strain
Purpose: Production of high-purity minicircle DNA devoid of bacterial backbone sequences from minicircle parental plasmids
Source: Developed by Dr. Zhiying Chen, Stanford University -6; Commercialized by System Biosciences (SBI) -3-9
Strain Background: Derived from BW27783 E. coli strain through multiple rounds of genetic engineering -6
Genotype:
Cp8.araE ΔendA bla.lacY A177C 3BAD.I-SceI 2BAD.ΦC31 4BAD.Φ31 4BAD.Φ31 -8

Genomic Modification Features:

1. ΦC31 Integrase Expression Cassettes: Multiple copies of L-arabinose-inducible ΦC31 integrase expression cassettes integrated at three genomic loci: 2 tandem copies at ΔendA locus, 4 copies at araD locus, and 4 copies at galK locus -6

2. I-SceI Endonuclease Expression Cassettes: 3 tandem copies of L-arabinose-inducible I-SceI endonuclease expression cassettes integrated at UMU locus -6

3. Arabinose Transport System:

   • Constitutively expressed araE gene driven by artificial promoter cp8 (from BW27783 strain) -6-8

   • lacY A177C mutant gene driven by bla promoter integrated at lacY locus, enhancing arabinose uptake -6-8

4. Other Modifications: ΔendA (deficiency in non-specific endonuclease I, improving plasmid quality) -8

Characteristics:

1. Minicircle Production Specificity: This strain is the only E. coli strain genetically engineered for efficient minicircle DNA production; other strains cannot be substituted -3-5-7

2. Inducible Expression System: Upon L-arabinose induction, simultaneously expresses ΦC31 integrase and I-SceI endonuclease -3-4

3. Production Mechanism:

   • ΦC31 integrase mediates recombination between attB and attP sites on minicircle parental plasmid, generating minicircle DNA and a backbone circle -2-4

   • I-SceI endonuclease cleaves I-SceI recognition sites on the backbone circle, leading to degradation by bacterial exonucleases -2-4

   • Ultimately, only minicircle DNA devoid of bacterial backbone sequences is retained -2-6

4. Production Efficiency: Produces purified minicircles in a time frame and quantity similar to routine plasmid DNA preparation -3-5

5. Product Purity: Contaminating DNA accounts for only 0.4%-1.5% after induction; minicircle DNA purity exceeds 98% -6

Production Protocol:

1. Transform minicircle parental plasmid (e.g., BioVector? pMC.BESPX) into this strain

2. Culture to appropriate density, add L-arabinose for 5-hour induction -1-4

3. Harvest cells, purify minicircle DNA using standard plasmid extraction methods -3-5

Culture Medium: BioVector? LB Medium (supplement with appropriate antibiotic to maintain parental plasmid)
Culture Temperature: 37°C
Inducer: L-arabinose (final concentration 0.01%-0.1%)
Resistance: Depends on transformed minicircle parental plasmid (typically kanamycin)
Product Format: Competent Cells / Glycerol Stock
Control Vector: BioVector? pMC.BESPX-CMV-luci Minicircle Parental Plasmid
Quality Control:

• Transformation Efficiency Validation: ≥1×10? cfu/μg pUC19

• Minicircle Production Efficiency Validation: Qualified minicircle DNA yield upon induction

• Sterility: Negative

• Functional Validation: Complete conversion of parental plasmid upon induction
  Storage and Shipment: Store and ship at -80°C or on dry ice; avoid repeated freeze-thaw cycles
  Purchase Restrictions: Available to not-for-profit researchers only -3-9; commercial users must purchase pre-made minicircle DNA products -3
  Biosafety Level: 1 (Operate in a Class II biological safety cabinet)
  For Research Use Only

Related Products:

• BioVector? pMC.BESPX Minicircle Producer Plasmid

• BioVector? pMC.BESPX-MCS2 Minicircle Producer Plasmid (with multiple cloning site)

• BioVector? pMC.BESPX-CMV-luci Luciferase Reporter Minicircle Parental Plasmid

• BioVector? ZYCY10P3S2T Competent Cells

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