Team:NYMU-Taipei/chassis

(In grams per liter)

●Succinic acid             0.74 g
●KH2PO4                    0.68 g
●Sodium nitrate            0.12 g
●*Wolfe's vitamin solution  10 ml(stock)
●*Wolfe’s mineral solution  5  ml(stock)
●Ferric quinate solution   2  ml(0.27 g of FeCl3 and 0.19 g of quinic acid in 100 ml water)
●L-cysteine                0.005%(Instead of sodium thioglycolate) 
●Yeast extract             0.01%
●Polypeptone               0.02%
 Adjust pH to 7.0 with NaOH

Strict anaerobic conditions have been thought to facilitate BMP (Bacterial Magnetic Particles) production in AMB-1 cells. To induce AMB-1 forming magnetosome, we have to control low O₂ concentration.

Electroporation So...how to insert our plasmid into the AMB-1?

After plasmid constructions were done, we need to transform them into AMB-1. Electroporation was performed with a Gene Pulser (Bio-Rad Laboratories, Richmond, California, USA), at a capacitance of 25 micro-F and a resistance of 200Ω, and 0.1-cm cuvettes. Electroporation allows cellular introduction of large highly charged molecules such as DNA which would never passively diffuse across the hydrophobic bilayer core. This phenomenon indicates that the mechanism is the creation of nm-scale water-filled holes in the membrane.

Procedure:

● Step1: Harvested and washed AMB-1 with 10 mM TES buffer containing 272 mM sucrose (pH 7.5).

● Step2: Resuspended AMB-1 in the same buffer at 10^9 cells/ml.

● Step3: Transferred to 500 micro-liter of MSGM supplemented with 20 mM Mg2+ and 
incubated at 27°C overnight with shaking at 100 rpm.

● Step4: Diluted in 5 ml of MSGM containing 0.7% agar.

● Step5: Plated on 1% agar in MSGM incubated under anaerobic conditions.

(supplied with ampicillin at 5 ug/ml or kanamycin at 2.5 ug/ml)

References