IGEM:Cambridge/2008/Notebook/Magnetic Bacteria/2008/07/25

Preparation for Plasmid Assembly

Genes mms6 and mamC

• Recovered mamC and mms6 plasmids from agarose gel using Zymoclean gel DNA recovery kit and followed standard protocols
• Set up restriction digestion of biobricked mms6 and mamC

Protocol of Plasmid Restriction Digestion:

• Set up 2 digestion reactions for mms6 and mamC respectively in EcoRI and PstI
• Reagents added in the following order:
  • 15μL SDW
  • 2μL 10x Fast Digest Buffer
  • 2μL DNA
  • 1μL EcoRI
  • 1μL PstI
• Tubes spun down and incubated for 10 min at 37°C
• Heat block at 80°C for 5 min to denature enzymes

Plasmid Vector pSB3K3

http://partsregistry.org/Part:pSB3K3

• Containing ccdB
• Kanamycin resistance

• Transform ccdB resistant E.coli strain DB3.1 with pSB3K3 extracted from filter paper
• Followed standard transformation protocol with the addition of CaCl2
• Cells plated out (neat and 1/10 dil.) onto agar plates with Kanamycin (25μg/mL)

Promoter I0500

http://partsregistry.org/Part:BBa_I0500

• Using biobrick DNA recovered from registry on 23/7/2008
• Do PCR with I0500, gel electrophoresis and recover DNA from gel using Gel DNA recovery kit
• Biobrick BBa_1006 of about 250bp used as reference
• DNA run on 0.8% agarose gel with SyBr Green

PCR protocol with 34 cycles:

• 5μL DNA
• 1μL of each primer VR and VF2
• 3μL SDW
• 10μL Finnzyme Master Mix
• Total volume of 20μL

Protocol for preparation of SyBr Green 0.8% Agarose Gel:

• 0.8g of Ultrapure agarose powder
• Add 100ml of TAE
• Microwave to dissolve agarose powder
• Add 100μL SyBr Green when bottle is cool enough to hold
• Gel should be about 6mm deep
• Run gel with 80V

Result:

• Bright band of about 1.2kb found on gel corresponding to I0500

Preparation of M9 Medium (Iron Free)

M9 salt and glucose solution is reduced in iron content using Chelex-100 resins, which are removed by filtration. Millipore filter used to pass the prepared M9 medium (glucose added) to sterilise the medium at the end. Sterilised deionised water should be used in the preparation of iron citrate stock.

Preparation of M9 Medium

5X M9 salts contain:

• 64 g Na2HPO4.7H2O
• 15 g KH2PO4
• 2.5 g NaCl
• 5 g NH4Cl

For the M9 medium,

Component Concentrations:

• 1x M9 salts
• 2mM MgSO4
• 0.1mM CaCl2
• In SDW

For 500ml of media:

• 250ml 2xM9 salts
• 10ml 0.1M MgSO4 (MgSO4•7H2O used so 0.493g added to 20ml H2O)
• 100μl 0.5M CaCl2 (0.055g anhydrous CaCl2 into 1000μl H2O)
• 234.9ml sterile deionized H2O
• 5ml 40% glucose (4g glucose in 6ml H2O)

Protocol:

• To make up a 5x M9 concentrate we need to add 56.4g in 1L H2O. Thus for 2xM9 we need to dissolve 11.28g M9 into 500ml H2O
• Disslove glucose into the M9 salt solution
• Pass M9 salts and glucose solution through 50ml Chelex-100 columns to reduce iron content of reagents before use
• Residual chelex 100 removed by filtration
• Dissolve other components into solution

Preparation of 200ml iron citrate stock (iron:citrate ratio = 1:100)

Reagents needed:

1. Tri-sodium citrate

CAS No: 68-04-2 Hazard: Irritation to skin, eyes and respiratory tract 400mM needed Dissolve 23.528g of Na3C6H5O7 sodium citrate into 200ml distilled deionized water

1. Ferrous sulfate FeSO4•7H2O

CAS No. 7720-78-7 Hazard: Irritation to skin, eyes and respiratory tract 4mM needed 0.222g of FeSO4•7H2O dissolved in 200ml of aqueous sodium citrate solution to give 4mM FeSO4

1. Aqueous sodium hydroxide

250ml of 0.5M NaOH needed to adjust stock to pH7 Dissolve 5g of NaOH into 250ml of distilled deionised water Hazard: corrosive

Ferrous sulfate should be added when needed in the experiment to prevent oxidation of Fe(II) to insoluble Fe(III) precipitates. Anaerobic condition needed when incubating E.coli to prevent such oxidation reaction which affects soluble iron ion concentration in solution and the iron uptake by bacteria.