A. WET LAB PROTOCOLS

I.Recipes

I.a Agarose gel (50ml of 1% gel)

1. Weight 0.5 g of agarose
2. Dissolve in 50 ml of 1x TAE buffer
3. Microwave until completely dissolved
4. Cool down to ~50^oC (doesn't burn when you hold it in your hand)
5. Add 5ul of SybrSafe
6. Pour the gel into the casting stand
7. Cover the casting stand with foil to protect it from light (SybrSafe is sensitive to light)
8. Wait for the gel to solidify
9. Load the samples (5-10ul of sample + 1-2ul of Loading Dye)
10. Load 7-10ul of 2-log ladder
11. Run the gel at 110 V for ~45 min
12. Use lower gel percentage for larger samples (plasmids etc.) and higher gel percentage for smaller samples (~200-400 bp PCR products)

I.b. Buffers

I.b.1. 50X TAE Buffer (1 liter)
1. 242 g Tris-base
2. 18.61 g EDTA
3. 57.1 ml glacial acetic acid
4. Water top up to 1L

I.b.2. 1X TAE Buffer (1 liter)
1. Measure 20 ml of 50X TAE buffer
2. Add 980 ml dH2O

I.c. Growth media

I.c.1. Liquid Broth (LB) Medium – 250ml
1. The following ingredients have been added to a 250ml bottle:
- 2.5 g Tryptone
- 1.25 g Yeast Extract
- 2.5 g NaCl
- 250ml dH2O
2. Loose the lid of the bottle
3. Secure the lid with autoclave tape
4. Autoclave
5. Store the media at 4ᴼC (i.e. in the fridge)

I.c.2. LB agar (250 ml ~ 10 plates)
1. Add the following ingredients in a 250ml bottle:
- 2.5 g Tryptone
- 1.25 g Yeast Extract
- 2.5 g NaCl
- 3.75 g agar
- 250ml dH2O
2. Loose the lid of the bottle
3. Secure the lid with autoclave tape
4. Autoclave
5. Pour plates next to a Bunsen burner (if add antibiotics to the LB agar, antibiotics should be added only when the temperature of the flask becomes below 60ᴼC)
6. Wrap plates in foil and store them at 4ᴼC (i.e. in the fridge)

I.c.3. Chrome azurol (CAS) plates agar
CAS agar was prepared with a mixture of 90 ml Y minimal agar and 10 ml CAS mix which were prepared and autoclaved separately (Schwyn and Neilands, 1987). Following cooling to ~45 °C, the mixtures were mixed slowly ( with a pipette) and swirled until homogenous.
Y minimal agar
1.The agar was prepared by dissolving the following ingredients in 50 ml pure water:
- 0.169 g of sodium glutamate (Sigma)
- 0.3 g Tris base (Fisher Scientific)
- 0.1 ml of MgSO4.7H20 (10 % w/v)
- 0.1 ml of CaCl2.6H2O (22 % w/v)
- 0.1 ml of K2HPO4.3H2O (22 % w/v)
2. pH was adjusted to 6.8
3. 1.5 g agar was then added to the medium and autoclaved
CAS mix
For preparation of 100 ml CAS mix, 10 ml of 1 mM FeCl₃.6H2O (dissolved in 10 mM HCl) and 60.5 mg of CAS powder were dissolved in 50 ml of pure water giving a final volume of 60 ml. 72.9 mg of hexadecyltrimethylammonium bromide (HDTMA) was dissolved in 40 ml pure water and both solutions were mixed with constant stirring giving a total of 100 ml CAS mix solution. This dark purple solution was autoclaved and stored at room temperature in the dark. Mixing 10 μl of this solution with 90 μl Y minimal agar would give a final concentration of 10 μM FeCl₃. To prepare a high iron solution, 10 ml of 6 mM FeCl₃.6H2O (dissolved in 10 mM HCl) was used making a 60 μM FeCl₃ deep blue CAS mix solution.

I.c.6. SOC medium (200ml)
1. Add 1g yeast extract
2. Add 4 g tryptone
3. Add 2ml of 1M NaCl
4. Add 0.4ml of 1M KCl
5. Add 4ml of 0.5M Mg SO₄
6. Add 2ml of 1M MgCl²
7. Top up with dH2O -up to 200ml
Adjust the pH to 7.5
8. Autoclave+ cool down
9. Add 4ml of 20% glucose solution

I.c.7. M9 + agar
1. Add 180ml dH2O
2. Add 20 ml M9 salts
3. Add 3g agar
4. Autoclave samples + cool down
5. Add 0.4 ml 1M MgSO⁴
6. Add 0.4 ml 100mM CaCl²
7. Add 2 ml 50% glucose
8. Add 0.1 ml thiamine (1% stock)
9. Add 0.2 ml chloramphenicol (50mg/ml)
10. Pour plates

I.c.8. Defined medium
Autoclavable ingredients:
-MOPS-8.3625g
-Tricine-0.7125g
-NH₄Cl-0.5g
-K₂SO₄-550µl (0.5M stock solution)
-CaCl₂-5µl (100mM stock solution)
-MgCl₂-525µl (1M stock solution)
-NaCl-5ml (1M stock solution)
-K₂HPO₄-2.6375ml (0.5 M stock solution)
-Casamino Acids-2g
-Trace metals-1ml (1000x stock solution)
-Distilled Water-981ml
Optional ingredients:
-FeCl3 600µl (10mM stock solution)
Non-autoclavable ingredients:
-Sterile Glucose 8ml (25% solution)
-Sterile Thiamine 1ml (1% solution)

II.Protocols for cloning

II.a. Introduction - strains used
Escherichia coli Top10
-naturally competent
-has streptomycin resistance
Escherichia coli DH5α
-naturally competent
-have several mutations that eliminate restriction endonucleases that might degrade plasmids
Escherichia coli BL21
-strain used for protein overexpression
-T7 expression system
Escherichia coli W3110
- wild-type strain (wild-type corresponding to the mutant JC28 strain)
Escherichia coli JC28
- mutant strain- lacks entC gene
-does not produce enterobactin

II.b. Introduction - plasmids used
pSB1C3
-iGEM plasmid for constitutive gene expression
-has RFP cloned into its multiple-cloning site
-selectable marker- chloramphenicol
pBSKII
-plasmid for constitutive gene expression
-selectable marker- ampicillin
pUC18
-high copy number plasmid
-selectable marker-ampicillin
pET15b
-plasmid used for gene overexpression
-selectable marker- ampicillin
-plasmid has a His Tag
pET28a
-plasmid used for gene overexpression
-selectable marker-kanamycin

III.c. Working concentrations for the antibiotics used
-Ampicillin (Amp)-100 µg/ml
-Chloramphenicol (Cm)-30 µg/ml
-Kanamycin (Kan)-50 µg/ml
-Streptomycin (Str)25 µg/ml

II.d. Making competent cells
Prepare:
*Cool in a freezer: Falcon tubes, CalC₂, CaCl₂-15% glycerol
*Cool in –80C: 15x Eppendorf tube for each strain

1. Inoculate single colony of each strain in 5-10 ml of LB (+Antibiotic) in 50 ml Falcon tube; Grow overnight at 37°C
2. Use 0.5 ml to inoculate in 50 ml LB (pH 7.2). Grow 2-4h to OD ~0.4-0.5
3. Transfer cells to ice cold 50ml Falcon
4. Cool the cells down on ice for 20 min and spin down at 4C, 5000 rpm for 10 min
5. Resuspend gently in 10 ml ice-cold 100mM CaCl₂
6. Incubate on ice for 20 min
7. Spin down as in step 4
8. Discard supernatant and resuspend cells in 5 ml ice-cold CaCl₂, cool for 10 min, spin down
9. Discard supernatant and resuspend cells in 1.5 ml ice-cold CaCl₂-15% glycerol
10. Aliquot 100ul into 1.5ml ice-cold Eppendorf tubes
11. Snap freeze and store in -80°C

II.e. Plasmid minipreps
0.Centrifuge 5-10 ml of overnight culture at maximum speed for 10 minutes
*** Overnight cultures for plasmid minipreps are set up by transferring a colony of bacteria in 10ml LB containing the corresponding antibiotic
1. Resuspend pellet in 250 µl resuspension buffer (P1 buffer)
2. Mix resuspension with 250µl lysis buffer (P2 buffer) and invert tubes 5 - 7 times
3. Add 350µl neutralization buffer (N3 buffer) and invert the tubes 5 - 7 times
4. Centrifuge samples for 5 min at 13000 rpm
5. Transfer supernatant to the provided spin column (take care not to disturb the pellet)
6. Centrifuge the column for 1 min at 12000 rpm
7. Discard the liquid that has run through the column (liquid waste)
8. Add 500 µl wash buffer (PE buffer)
9. Centrifuge samples for 1 min at 13000 rpm
10. Discard the liquid that has run through the column (liquid waste)
11. Repeat again steps 9, 10 and 11
12. Perform one additional centrifugation step (without adding any buffer) for removing residual ethanol
13. Transferred the dried column to a fresh Eppendorf tube
14. Add with 35µl elution buffer in the center of the column membrane
15. Incubate the columns with elution buffer for 2 min at room temperature
16. Elute plasmids by centrifugation for 2 min at 13000 rpm
17. Store samples in the freezer (-20°C)

II.f. Transformation

II.g. Ligation

II.h. Genomic DNA extraction

II.i. Genomic DNA extraction

II.j. PCR clean-up (DNA Purification by Centrifugation)

II.k. Nano-Drop (estimating concentration and purity of DNA samples)