User:Howard Boland/Notebook/Art from Synthetic Biology/2011/02/02

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BBa_I13504+BBa_katE

The following steps were performed towards a new composited part

Check overnight growth (BBa_I13504)

Growth worked fine - all three tubes grew.

Take Glycerol Stock (BBa_I13504)

For each tubes make glycerol stock

  1. 700µl Bacteria Broth
  2. 300µl 50% Glycerol

Miniprep samples(BBa_I13504)

Purify plasmids using QIAGen Miniprep kit

  1. Eluted in 35µl Water

Digestion

4 Digestions where setup

  1. BBa_katE - the pcr product was digested with EcoRI and SpeI (Upstream)
  2. BBa_I13504 was digested with XbaI and PstI

Gel

The digestion was run on a 2% Gel. Check against BBa_I13504 sample

  1. Lane 1: 100bp ladder
  2. Lane 2: Blank
  3. Lane 3: 50µl digested sample BBa_katE- E/S (~505bp)
  4. Lane 4: Blank
  5. Lane 5: 50µl digested sample BBa_katE- E/S (~505bp)
  6. Lane 6: Blank
  7. Lane 7: 50µl digested sample 1 BBa_I13504- X/P (875bp)
  8. Lane 8: 50µl digested sample 2 BBa_I13504- X/P (875bp)

02022011-double digests.jpg

5x M9 Salt, 200ml

Minimal media M9 salt, at pH7

  1. 1.2g di-Sodium hydrogen orthophosphate (Na2HPO4, 6g/l)
  2. 0.6g Potassium dihydrogen phosphate (KH2PO4, 3g/l)
  3. 0.1g NaCl (0.5g/l)
  4. 0.2g Ammonium chloride (NH4Cl, 1g/l)

Autoclave at 121ºC for 15 minutes

M9 Media, 200ml

  1. 150ml H2O, Distilled (Cooled to <50ºC)
  2. 40ml 5x M9 Salt
  3. 200µl 1M MgSO4 (1ml/l)
  4. 0.2g Casamino acids (1g/l)
  5. 20µl 1M CaCl2 (0.1ml/l)
  6. Adjust volume to 200ml using sterile H2O

Carbon Sources

  1. 1% Glucose Or 0.5% Glycerol

5 bottles, 10ml 50% Glycerol

  1. Add 5ml Glycerol
  2. Add 5ml water

Autoclave 20min in liquid cycle

Effector

  1. 3.6mM IPTG
  2. 5.3mM cAMP

0.1M CaCl2, 500ml

Used to prepare competent cells.

CaClx6H2O has a molecular weight of FW 219.08.

  1. To make 1M in one litre of water add 219.08g powder and increase volume by adding water to 1litre.
  2. To make 0.1M in 200ml of water add 0.1*(219.08g)/(1000ml/500ml) = 0.1*219.08g / 2 = 10.95g powder and increase volume by adding water up to 500ml.

Autoclave at 121ºC for 15minutes

0.1M MgCl2, 500ml

Used to prepare competent cells.

MgCl2x6H2O has a molecular weight of FW 203.3.

  1. To make 1M in one litre of water add 203.3g powder and increase volume by adding water to 1litre.
  2. To make 0.1M in 200ml of water add 0.1*(203.3g)/(1000ml/500ml) = 0.1*203.3g / 2 = 10.16g powder and increase volume by adding water up to 500ml.

Autoclave at 121ºC for 15minutes

2x 500ml water

Autoclave at 121ºC for 15minutes

Locate Potassium L-lactate

I am currently missing this substrate to grow my nanomagentic bacteria.

Test FSM with Pyruvic as Carbon Source

Since I have not ben able to locate lactate I am testing pryuvic acid as a carbon source.

To 200ml FSM I added 0.47g powder

  • pryuvic acids = 88.06 g/mol
  • 27mM in 200ml => (0.027)*88.06g/(1000ml/200ml)=0.47g