Team:Paris Bettencourt/Notebook/Trojan Horse/Tuesday 23rd July.html

Trojan Horse

ASDF

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Infectiveness characterization experiments

• The night before: prepare O/N of F+ cells, prepare O/N of phagemid producing cells (in this case : sT007 )
• Centrifugate phagemid producing cells
• Filter the supernatant 0.45um, stock it at 4°C (it contains the phages)
• Dilute 200x MGZ1, F+ from O/N
• Wait until OD600 = 0.7
• Immediately mix MGZ1 and the supernatant in different proportions
  
  

Here we tried 1/1 (vol supernatant/vol cells), 1/10 and 1/100

Immediately dilute each of them to 10-3, 10-4, 10-5, 10-6
• Plate 100uL of those dilutions on LB-agar, AMP, KAN, AMP+KAN
• Incubate O/N
• Count colonies
• Interpretation: Colonies on AMP: infected by litmus28i
  Colonies on KAN: infected by M13K07
  Colonies on AMP+KAN: coinfected by litmus28i and M13K07 phagemid producing cells
  

Conclusions:

• Cells that were mixed with 1/1 supernatant didn't survive the excess of antibiotics that were present in the supernatant.
• Even at low ratio like 1/100 the infection rate of litmus28i-GFP is about 100%. GFP detection confirmed this result: almost 100% of cells were fluorescent.
• Co-infection did occur. It would be interesting to test if those colonies are indeed producing phages.

Start O/N culture of M13K07 Start O/N culture of MGZ1,F+