Brown:Protocols:Magnetotactic Bacteria

From 2006.igem.org

I just threw this in here, but it needs- detabling as the wiki doesn't like word tables. --Johncumbers 17:53, 23 June 2006 (EDT)

iGEM Protocols: Edited by John Cumbers, June 23rd 06

Magnetospirillum sp. AMB-1 Bacterial growth medium preparation

These protocol are for the producing the growth medium needed by the magnetotactic bacteria.

1. Protocol from Dennis Bazylinski in Iowa State University (Prof. Palmore’s contact there) 2. (Komeili group)

Magnetospirillum magnetotacticum (aerobic) List of ingredients

1. Protocol from Dennis Bazylinski in Iowa State University (Prof. Palmore’s contact there)

Name Amount Place Note Price Wolfe’s Vitamin solution 10ml ATCC $34 5X10ml Wolfe’s Mineral Solution 5.0ml See Below KH2PO4 0.68g Palmore Lab Sodium succinate *6H2O 0.848g Chemical Stock Room May want to get new Product number: S2378-100g Price: $18.70

Sodium Tartrate*2H2O 0.575g Chemical Stock Room May want to get new Product number: 34696-25G-R Price: $18.20 Sodium acetate *3H2O 0.083g Palmore Lab 0.1% Resazurin 0.45ml Product number: 199303- 1G Price:$14.50 NaNO3 0.17g Palmore Lab, Chemical Stock Room Ascorbic acid 0.035g Palmore Lab NaOH A bit Palmore Lab Quinic Acid 0.19g Product name: (1R,3R,4R,5R)-(−)-Quinic acid Product number: 138622-25G Price: $40.70 FeCl3*6H2O 0.27g 97%-250g-$28 93-2606

After we make the liquid medium, we dispense 55 ml into 155 ml serum vials that have butyl rubber stoppers and crimp tops (vials are described as 125 ml in catalogues but they actually hold 155 ml). Bubble media for 0.5 h with N2 and then seal bottles and then sparge headspace for another 0.5 h with N2. Then autoclave. M.magneticum will grow and produce magnetosomes under anaerobic conditions. M. magnetotacticum will grow somewhat but needs 1 ml of sterile O2 injected into vial first for it to make maximum number of magnetosomes.

That is why we would send you magneticum. Medium should be colorless or close to that.

2. Magnetospirillum magneticum sp. AMB-1 (Komeili group) (anaerobic) John’s contact with Arash Komeili in UCBerkeley

Name Amount Place Note Price Wolfe’s mineral solution (without iron) 5ml See below KH2PO4 0.68g Palmore Lab NaNO3 0.12g Palmore Lab Sodium acetate 0.07g Palmore Lab Ascorbic acid 0.035g Palmore Lab Tartaric acid 0.37g Name: D-(-)-Tartaric Acid Product number: 95320-10G Price: $14.40

Name: DL Tartaric Acid Product number:T400- 25G Price: $7.60

Name: L-(+)-Tartaric Acid Product number: T109-100G Price: $22.90 Succinic acid 0.37g Product number: S9512-100G Price: $15.80 Sodium thiosulfate 0.05g Product number: 72049-250G Price: $20.40 Wolfe’s vitamin solution ATCC Ferric malate 100ul x 3mM Oxalic acid For washing Sigma-50g-15.20-75690 Kanamycin Stockroom? For selection Name: Kanamycin sulfate from Streptomyces kanamyceticus Product number: K1377-1G Price: $20.52

Other: N2 (gas tank)

- The medium is bubbled with N2(g) before autoclaving. - 1-5ml of air is added to each tube (~1-5% Oxygen) - Incubation at 30oC. - For agar plating, 7g of Agar for 1L of media. (vitamins, ferric malate and kanamycin added just at end) - Plates incubated in jars with 2-3% oxygen. - 5-7days until colonies appear.

Modified Wolfe’s Mineral solution: Name Amount Place Note Price Nitrilotriacetic acid (NTA) 1.5g Palmore Lab (KG) MgSO4*7H2O 3.0g Palmore Lab (HK) MnSO4*H2O 0.5g Sigma-100g-10.80 Cell culture grade NaCl 1.0g Palmore Lab FeSO4*7H2O 0.1g Palmore Lab Old 99+%-250g-23.00 93-2639 CoSO4*7H2O 0.1g Chemical Stock Room Old 98+%-100g-$15 93-2749 CaCl2*2H2O 0.1g Palmore Lab, chemical Stock room, Hayato ZnSO4*7H2O 0.1g Palmore Lab Sigma-100G-$21 Cell culture grade Z-0251 CuSO4*5H2O 0.025g Palmore Lab (SY) AlK(SO4)2*12H2O 0.01g Chemical Stock room Sigma-100G-$29.4 Cell culture grade A-6435 H3BO3 0.01g Palmore Lab (SY) Na2MoO4*2H2O 0.4g Chemical Stock Room Sigma-100G-23.40 Cell culture grade M1003 NiCl2*6H2O 0.01g Chemical Stock room old 99.9%-250g-$33 93-2810 99.999%-10g-$36 28-1160