BioVector® p112A1NE High-Copy Yeast Expression and Functional Complementation Vector / p112A1NE 高拷贝酵母表达与功能互补鉴定质粒

I Product General Information and Molecular Background

• Plasmid Nomenclature: p112A1NE (Alternative nomenclature: p112AINE).

• Vector Type and Target Systems: Yeast High-Expression Shuttle Vector / cDNA Library Construction Backbone.

  Originally developed by Frommer, Willmitzer, and Riesmeier (published in The EMBO Journal, 1992), p112A1NE is a classic, high-performance yeast expression vector widely used in plant physiology, cell biology, and transporter genetics. Its main historical and current application is the functional complementation and heterologous characterization of membrane transport proteins (such as sucrose carriers, hexose/sugar porters, phosphate transporters, and sulfate transporters) inside nutrient-uptake deficient mutant strains of Saccharomyces cerevisiae.

• Core Genetic Architecture & Promoter Drive:

  • Promoter (Expression Drive): Driven by the robust, truncated yeast $ADH1$ (Alcohol dehydrogenase 1) constitutive promoter. This ensures sustained, high-level transcription of cloned open reading frames (ORFs) or cDNA libraries across all growth phases without requiring chemical inducing agents like galactose or copper.

  • Auxotrophic Selection Marker: Utilizes the yeast $TRP1$ gene encoding phosphoribosylanthranilate isomerase. This allows for positive selection in tryptophan-auxotrophic host yeast strains (e.g., mutant lines growing on Synthetic Defined media lacking tryptophan, SD-Trp).

  • Replication Origins:

    • Yeast Phase: Powered by the $2\mu$ origin ($2\mu\text{ ori}$), ensuring high-copy autonomous plasmid replication and maintenance ($10 - 40$ copies per cell) inside the yeast cytoplasm.

    • Bacterial Phase: Powered by a standard pBR322 origin (\textit{ori}), supporting stable high-copy amplification inside cloning strains of Escherichia coli.

• Cloning coordinates and Cloning Selection:

  • Bacterial Selection Marker: Ampicillin resistance ($Amp^R$), mediated by the $\beta$-lactamase gene (bla), selected at a standard $100\ \mu\text{g/mL}$ working dose.

  • Cloning Matrix: Features a specialized Multiple Cloning Site (MCS) flanked by universal priming sites (e.g., M13 forward/reverse parameters). It is ideal for inserting single plant/fungal transporter cDNAs or staging whole-genome directionally-cloned cDNA libraries via traditional restriction enzyme digestion.

  • Total Vector Size: Approximately 5,725 bp (empty backbone configuration).

II Strategic Research Value and Membrane Transporter Complementation

The p112A1NE vector serves as a critical screening platform in plant and microbial biology due to its high-copy nature and strong constitutive expression:

1. Heterologous Functional Characterization of Plant Transporters:

   Many plant membrane proteins (e.g., wheat phosphate transporters TaPHT1/TaPT3-2D, apple sulfate transporters MdSultr, or spinach sucrose carriers) cannot be accurately characterized biochemically in artificial membranes. By cloning these eukaryotic transporter ORFs into p112A1NE and converting them into specialized yeast mutants, researchers can easily calculate substrate affinity ($K_m$), transport kinetics ($V_{max}$), and H\textsuperscript{+}-coupled pH dependencies.

2. Yeast Mutant Complementation Assays:

   • Phosphate Transporter Mapping: Transformed into the yeast $P_i$ uptake-defective mutant MB192 (or similar strains lacking endogenous high-affinity phosphate transporters). Growth of the p112A1NE-transporter strain on low-phosphate ($20\ \mu\text{M}\ P_i$) media confirms the gene's function as a high-affinity phosphate transporter.

   • Sugar Porter Mapping: Transformed into hexose-transport-deficient yeast (e.g., the EBY.VW4000 strain) to evaluate fungal or plant sugar porters (PiHXT5/PiST12), checking their capability to rescue yeast growth using specific carbon sources like D-glucose, D-fructose, or D-xylose.

3. Heavy Metal Detoxification and Tolerance Assays:

   p112A1NE is frequently used to overexpress novel cysteine-rich peptides or metallothioneins to screen for cellular heavy metal resistance, measuring survival shifts under Cadmium ($Cd^{2+}$) or Zinc ($Zn^{2+}$) stress.

III Laboratory E. coli Propagation, Quality Control, and Yeast Transformation Protocols

1. Plasmid Amplification and Isolation in Escherichia coli

• Recommended Competent Host Strain: Escherichia coli DH5$\alpha$ or Top10 cloning-grade cells.

• Bacterial Selective Pressure: Supplement standard LB broth or agar matrices with $100\ \mu\text{g/mL}$ Ampicillin.

• Amplification Protocol:

  1. Introduce 1 $\mu$L of p112A1NE plasmid DNA into 50 $\mu$L of competent DH5$\alpha$ cells.

  2. Incubate on ice for 30 minutes, heat-shock at 42 °C for 45 seconds, and place back on ice for 2 minutes.

  3. Supplement with 250 $\mu$L of SOC or LB broth; recover at 37 °C with shaking at 220 rpm for 45 minutes.

  4. Spread the suspension onto ampicillin-selective LB agar plates and incubate inverted at 37 °C for 14 – 16 hours.

  5. Pick an isolated colony into 5 – 10 mL of selective LB broth, grow at 37 °C with shaking at 250 rpm overnight, and isolate high-purity plasmid DNA using a standard silica-column miniprep kit.

2. Analytical Quality Control and Verification Mapping

Prior to attempting yeast transformations or downstream functional assays, strictly audit the quality and identity of the isolated plasmid DNA:

• Spectrophotometric Metrics: Measure purity using a NanoDrop instrument. Quality Control Limit: The $OD_{260}/OD_{280}$ ratio must fall strictly within the 1.8 – 2.0 range. Ensure the preparation is free from residual bacterial genomic DNA, protein fractions, or ethanol carryover.

• Restriction Enzyme Verification Mapping:

  Digest the empty p112A1NE backbone or the recombinant construct using diagnostic restriction enzymes (e.g., enzymes targeting the cloning site boundaries). Run the fragments on a 1% agarose gel. Quality Control Band Profile: The empty backbone must resolve as a single clear band at approximately 5,725 bp upon linearization. Recombinant vectors should clearly yield a 5.7 kb backbone band plus the distinct insert fragment.

• Recommended Sequencing Primers:

  • M13/pUC Forward Primer: 5'-gttttcccagtcacgac-3'

  • M13/pUC Reverse Primer: 5'-caggaaacagctatgacc-3'

3. Lithium Acetate (LiAc/PEG-3350) Small-Scale Yeast Transformation Matrix

To transform p112A1NE into auxotrophic mutant yeast hosts (e.g., MB192 or EBY.VW4000), execute the standard Lithium Acetate protocol:

1. Yeast Starter Culture: Inoculate the target auxotrophic mutant yeast strain into appropriate permissive medium (e.g., YPD supplemented with 2% maltose for sugar mutants, or standard YPD for phosphate mutants) and grow at 30 °C with shaking overnight.

2. Log-Phase Harvesting: Back-dilute the starter culture into fresh permissive medium to an $OD_{600}$ of approximately 0.2. Incubate at 30 °C with shaking until the culture reaches an $OD_{600}$ of 0.6 – 0.8 (the peak window for transformation competence).

3. Conditioning and Washing: Harvest the yeast biomass by centrifuging at 3,000 rpm (~1,000 g) for 5 minutes. Wash the cell pellet with 25 mL of sterile deionized water, centrifuge again, and resuspend the cells in 1 mL of freshly prepared 0.1 M LiAc solution. Transfer the slurry to a sterile 1.5 mL tube, spin at maximum speed for 20 seconds, and completely aspirate the liquid.

4. Assembly of the Transformation Matrix: Add the following reagents to the transformation pellet strictly in the exact sequence listed:

   • 240 $\mu$L of sterile 50% w/v PEG 3350 solution

   • 36 $\mu$L of sterile 1.0 M LiAc solution

   • 25 $\mu$L of high-concentration Single-Stranded Carrier DNA (e.g., Salmon Sperm DNA, 2 mg/mL, pre-boiled at 95 °C for 5 minutes and immediately chilled on ice)

   • 5 – 10 $\mu$L of purified p112A1NE plasmid DNA (equivalent to 1 – 2 $\mu$g)

5. Homogenization: Mix the viscous mixture gently using a pipette tip or light vortexing until the yeast pellet is thoroughly resuspended. Incubate statically at 30 °C for 30 minutes.

6. Heat Shock Application: Transfer the transformation tubes directly into a 42 °C water bath and incubate for exactly 15 – 20 minutes. (Optimize incubation time depending on the fragility of specific transporter mutant strains).

7. Pelleting and Plating: Centrifuge at 12,000 rpm for 30 seconds and carefully remove the PEG/LiAc supernatant. Resuspend the cell pellet gently in 100 – 200 $\mu$L of sterile water or sterile saline solution.

8. Auxotrophic Selection: Spread the cell suspension onto solid Synthetic Defined (SD) dropout media lacking tryptophan (SD-Trp), supplemented with the appropriate alternative nutrients required by the specific mutant line.

9. Incubation: Incubate the plates inverted at 30 °C for 3 – 5 days until distinct, well-isolated $TRP1^+$ prototrophic transformant colonies emerge.

4. Functional Complementation and Kinetic Assay Guidelines

• Baseline Controls: Always carry three parallel control strains during functional validation: Wild-type yeast, the mutant strain transformed with the empty p112A1NE vector, and the mutant strain transformed with the recombinant p112A1NE-Transporter construct.

• Assay Environment Tuning: Because most plant and fungal transport processes are driven by a proton gradient ($H^+$-symporters), verify the pH of the assay medium. For most p112A1NE-driven plant phosphate and sugar transporters, adjust the liquid YNB assay medium to an optimal pH of 5.5 – 6.0. Monitor cell growth by tracking the $OD_{600}$ kinetic profiles every 6 hours over a 24-hour period to confirm that the cloned gene successfully restores normal nutrient uptake.

5. Long-Term Cryopreservation Parameters

• Cryoprotectant Freezing Mix: 70% sterile YPD or SD-Trp liquid broth blended with 30% sterile analytical-grade Glycerol (yielding a final glycerol concentration of 15% v/v).

• Storage Workflow:

  1. Scrape healthy, fresh yeast transformant colonies from an SD-Trp plate or harvest a mid-log phase liquid culture.

  2. Suspend the cells thoroughly in 1 mL of the sterile glycerol cryoprotectant mix inside a sterile cryogenic vial.

  3. Mix by inversion, then transfer the vials directly into a -80 °C ultra-low freezer for long-term archiving. Avoid frequent temperature cycles to maintain cell viability and ensure consistent transporter expression levels upon thawing.

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