Difference between revisions of "Team:Aix-Marseille/Amelioration part:BBa K1445000"

 
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{{Aix-Marseille|title=M13 Amelioration part|toc=__TOC__}}
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{{Aix-Marseille|title=M13 part improvement|toc=__NOTOC__}}
  
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As we wanted to make phage like particles, we needed to use a [[Team:Aix-Marseille/M13_Design#Phagemid|M13 phagemid]].
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Initially we tried using [http://parts.igem.org/Part:BBa_K1445000 BBa_K1445000].
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After a transforming TG1 with the biobrick, we selected colonies by the use of chloramphenicol, and infected them with helper phage (M13KO7).
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We then purified the [[Team:Aix-Marseille/Experiments/Protocols#Production_of_large_quantities_of_helper_phage|phage]] containing the phagemid and infected ''E.coli'' GM1 bacteria with the resulting preparation.
  
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After overnight growth, we counted the colonies.
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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):
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{|
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|
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|colspan="3" style="text-align: center"|pBlueScript II
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|
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|colspan="3" style="text-align: center"|BBa_K1445000
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|-
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|
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|Ampicilin
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|Kanamycin
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|Ampicilin + Kanalycin
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|
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|Chloramphenicol
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|Kanamycin
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|Chloramphenicol + Kanamycin
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|-
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|1µL
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|162
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|606
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|1
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|1µL
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|0
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|957
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|0
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|-
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|10⁻²
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|1
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|33
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|0
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|10⁻²
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|0
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|186
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|0
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|-
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|10⁻³
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|0
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|1
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|0
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|10⁻³
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|0
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|28
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|0
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|-
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|500µL
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|uncountable
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|uncountable
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|7
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|500µL
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|0
 +
|uncountable
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|0
 +
|}
  
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As we use a phage like particle, we needed to use the [https://2017.igem.org/Team:Aix-Marseille/M13_Design#Phagemid M13 phagemid].
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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.
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Results weren’t good, after a transformation using TG1 bacteria with the [http://parts.igem.org/Part:BBa_K1445000 BBa_K1445000], we picked up the colonies on a chloramphenicol plate and infected them with our M13KO7 phage like particle.  
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That means that the phagemid, which carried chloramphanicol resistance, was not packaged at all.
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With the pBlueScript as a control, we observe ampicilin resistance acquired by infected showing packaging, albeit at a lower level of bacteria than expected.
  
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After that, we executed the [https://2017.igem.org/Team:Aix-Marseille/Experiments/Protocols#Production_of_large_quantities_of_helper_phage Large quantities of helper phage] protocol and infected GM1 bacteria with the resulting phage like particle.
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The low levels of phagemid packaging observed, considerably lower than expected, could arrise from a number of causes:
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* Plasmid loss in part of the population;
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* Poor packaging due to bad recognition of the M13 origin, as described on our [[Team:Aix-Marseille/Model|model]]
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* Lower infectivity if PLP
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* Poor isolation of PLP by our protocol.
  
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After an overnight grow, we counted the colonies.
+
A number of recombination events are also observed with bacteria simultaneously acquiring two different resistances.
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Here are the results for the M13 from the BBa_K1445000, and the pBlueScript II plasmid that we used:
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[[File: M13 biobrick tableau.png|1000px|right|thumb|]]
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In this experiment, for better results, everything was done three times, and these results are averages.
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Latest revision as of 01:42, 2 November 2017

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.