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Team:Paris Bettencourt/Notebook/Trojan Horse/Tuesday 23rd July.html
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<a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Trojan_Horse" target="_blank" class="tbnotelogo THlogo"> ASDF </a> | <a href="https://2013.igem.org/Team:Paris_Bettencourt/Project/Trojan_Horse" target="_blank" class="tbnotelogo THlogo"> ASDF </a> | ||
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Colonies on AMP+KAN: coinfected by litmus28i and M13K07 phagemid producing cells<br /> | Colonies on AMP+KAN: coinfected by litmus28i and M13K07 phagemid producing cells<br /> | ||
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Latest revision as of 17:52, 3 October 2013
Trojan Horse
ASDF23th July
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
- 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
Results
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+
