M13 part improvement

As we wanted to make phage like particles, we needed to use a M13 phagemid. Initially we tried using [http://parts.igem.org/Part:BBa_K1445000 BBa_K1445000]. After a transforming TG1 with the biobrick, we selected colonies by the use of chloramphenicol, and infected them with helper phage (M13KO7). We then purified the phage containing the phagemid and infected E.coli GM1 bacteria with the resulting preparation.

After overnight growth, we counted the colonies. Here are the results for the M13 from the BBa_K1445000 and the pBlueScript II plasmid that we used (all measurements were made in triplicate):

	pBlueScript II				BBa_K1445000
	Ampicilin	Kanamycin	Ampicilin + Kanalycin		Chloramphenicol	Kanamycin	Chloramphenicol + Kanamycin
1µL	162	606	1	1µL	0	957	0
10⁻²	1	33	0	10⁻²	0	186	0
10⁻³	0	1	0	10⁻³	0	28	0
500µL	uncountable	uncountable	7	500µL	0	uncountable	0

It is immediately apparent from this table that with the BBa_K1445000 biobrick the only antibiotic resistance we recover in phage and PLP is kanamycin resistance carried by M13KO7. That means that the phagemid, which carried chloramphanicol resistance, was not packaged at all. With the pBlueScript as a control, we observe ampicilin resistance acquired by infected showing packaging, albeit at a lower level of bacteria than expected.

The low levels of phagemid packaging observed, considerably lower than expected, could arrise from a number of causes:

Plasmid loss in part of the population;
Poor packaging due to bad recognition of the M13 origin, as described on our model
Lower infectivity if PLP
Poor isolation of PLP by our protocol.

A number of recombination events are also observed with bacteria simultaneously acquiring two different resistances.

Team:Aix-Marseille/Amelioration part:BBa K1445000

M13 part improvement