Hi, my team and I are currently performing experiments applying phage display technology with the E.Coli SS320 strain and Helper Phage (M13K07) to produce small peptides.
After we transformed the phagemid bearing the F1 origin into SS320 and perform phage production, we moved on to quantify the number of phages attained after purification. We use the following materials (along with details):
+ Ecoli SS320 (no phagemids, only F' sustained with tetracycline)
+ LB Miller medium (as well as agar)
+ Bottom agar concentration: 15g/l, Top agar concentration: 7,5g/l
+ Infection rate: 500ul bacterial culture + 100 ul phage dilution in PBS
+ Infection time (before mixing with melted Top agar): 1h and then leave the plates at 37oC overnight
However, after the night, no visible plaques are recognized on all of the plates. Therefore we have many questions regarding our current protocol:
1> Is it necessary for the helper phage to infect the SS320 transformed with the phagemid?
2> Do we need to use minimum culture such as M9 to sustain the male SS320 (those who have pili)?
3> Do we have to change to either LB Lennox or Luria to decrease the NaCl concentration?
4> Is it necessary to perform IPTG-induced translation to differentiate the surviving SS320?
It would be a great help for us to have all the questions answered. If you have any other advice, please let us know. Thank you all.
If you grow SS320 with tetracycline there is no need to grow in M9 minimal. That is used for some strains where the selection is on minimal to maintain the F', but it is not the case for SS320.
I doubt it has anything to do with LB Lennox or Luria. I have used both.
It could be that you don't have the right dilution so you can't see individual plaques, M13 can grow to very very high titer (I've had more than 10e12 PFU/ml sometimes so be sure you are doing a full range of dilutions.
Also M13 makes fairly weak plaques and M13KO7 even more so expect to look for tiny plaques.
Lastly you are adding a lot of cells. I would use 50-100ul of culture per plate (and not 500). The more cells you have the fewer replication cycles they go through and therefore the smaller and fainter plaque you would get.
Alternatively you can also titer M13KO7 by transduction and not by plaque formation. You can do the same dilution series you would do for looking at isolated plaques but then plate on kanamycin plates and count colonies.
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