PRODUCT

USAGE

SUGGESTED SUPPLIER

Molecular Cloning

NcoI

Linearize PandaPure Expression Vector (pPPC-vector)

NEB, cat. R3193V

Gibson Assembly kit

Insert coding sequence into pPPC-vector by homology

NEB, cat. E5520S

Expression

BL21(DE3) competent cell

High-efficient competent cells of an expression strain. Other strains are also compatible, such as BL21AI, if it has:
- T7 RNA Polymerase expression;
- no kanamycin or chloramphenicol resistances.

Invitrogen, cat. C601003

SOC medium

Seed culture, to avoid potential toxicity from leaky expression

Sigma-Aldrich, cat. S1797

IPTG

Induce T7 system for over-expression of proteins

Sigma-Aldrich, cat. I6758

Anhydrotetracycline (aTc)

Induce organelle construction via tet promoter

Sigma-Aldrich, cat. 37919

Purification

Bacterial Lysis Reagent *

Non-ionic detergent for gentle, efficient disruption of bacterial cells without mechanic forces, removing cytosolic impurities.

Thermo Scientific, cat. 78243
Millipore, cat. 70584

Lysozyme (optional)

Disrupt bacterial cell walls, removing large-size impurities.

Sigma-Aldrich, cat. 1.05281

Common Consumables and Labwares

Pipettes and tips

For microliter operations

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Incubator and shaker

For Gibson assembly reaction (at 50°C)
For bacterial growth on agar plate (at 37°C)
For liquid culture of bacteria at lab-scale flask (at 37°C, 220 rpm), or deep-well plates (at 37°C, 900 rpm).

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LB broth and agar, Miller

For bacterial growth in transformation, propagation and protein expression.

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Kanamycin, 50 mg/ml
Chloramphenicol, 30 mg/ml

Antibiotics for PandaPure Expression Plasmid, and Organelle Plasmid, respectively.

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Culture tube,
shake flask,
and deep-well plates

For bacterial culture at 1-5 ml (tube), for propagation and trial;
25 ml - 1L (flask), for Lesser Panda in lab-scale production;
and 100 μl - 1 ml (plates), for Hi Panda in screening assays.

Tube: Nunc, cat. 11337704
Flask: NEST, cat. 782101
Plate: Nunc, cat. 278743

Centrifuge
and consumables

To separate bacterial cell, organelles by centrifugation:
1.5 or 2 ml centrifuge tube, for trial tests;
50 ml or large falcon tube, for Lesser Panda production;
2-ml deep-well plates, for Hi Panda in screening assays.

Eppendorf 5810
Rotor FA-45-48-11
Rotor FA-45-6-30
Plate Rotor A-2-DWP-AT

0.22 μm filter (optional)

To remove remained organelle pieces by filtration

Millipore, cat. GSWP04700