Team:SDU-Denmark/project-p

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(Stability assay)
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===Stability assay===
===Stability assay===
To dertermine the stability of our pSB1C3-K343004 plasmid, a stability experiment was carried out according to protocol[[https://2010.igem.org/Team:SDU-Denmark/protocols#Stability_assay SA1.1]]. ''E.coli'' MG1655/pSB1C3-K343004 was grown in LB media without chloramphenicol, whereby no selection pressure is excerted on the bacteria. Dilutions of the culture was spreaded on LA plates and LA plates with 35ug/mL chloramphenicol, respectively, and the colony forming units (cfu) was determined for each plate. The cfu for the LA plates represents the total amount of bacteria in the culture, and the cfu of LA plates with chloraphenicol corresponds to the amount of plasmid carrying bacteria. The percentage of the total amount of bacteria carrying the plasmid was plotted in a semi-logarithmic graph as a function of number of generation.<br><br>
To dertermine the stability of our pSB1C3-K343004 plasmid, a stability experiment was carried out according to protocol[[https://2010.igem.org/Team:SDU-Denmark/protocols#Stability_assay SA1.1]]. ''E.coli'' MG1655/pSB1C3-K343004 was grown in LB media without chloramphenicol, whereby no selection pressure is excerted on the bacteria. Dilutions of the culture was spreaded on LA plates and LA plates with 35ug/mL chloramphenicol, respectively, and the colony forming units (cfu) was determined for each plate. The cfu for the LA plates represents the total amount of bacteria in the culture, and the cfu of LA plates with chloraphenicol corresponds to the amount of plasmid carrying bacteria. The percentage of the total amount of bacteria carrying the plasmid was plotted in a semi-logarithmic graph as a function of number of generation.<br><br>
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[[Image:Team-SDU-DC stab 2.png|400px|center]]<br>
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[[Image:Team-SDU-DC stab 2.png|thumb|400px|center| Stability assay of ''E. coli'' strain MG1655-pSB1C3-K343004 showing that almost all cells have shed their plasmids within 20 generations, however some cells kept the plasmids for up to 40 generations. All data can be seen under [https://static.igem.org/mediawiki/2010/0/09/Team-SDU-_flhDC_stability_assay.ZIP Raw data]]]
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All data can be seen under [https://static.igem.org/mediawiki/2010/0/09/Team-SDU-_flhDC_stability_assay.ZIP Raw data]<br><br>
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<br><br>
As seen in the graph, almost all of the bacteria had shedded the plasmid after 20 generations, suggesting that the plasmid is only stable within the cell for a few generations (<20). This is presumably due to the strain brought upon the bacteria by the plasmid. Thereby when the bacteria are carrying a high-copy plasmid like pSB1C3-K343004 it is to expect that the bacteria will quickly shed the plasmid when no longer exposed to a selection pressure.
As seen in the graph, almost all of the bacteria had shedded the plasmid after 20 generations, suggesting that the plasmid is only stable within the cell for a few generations (<20). This is presumably due to the strain brought upon the bacteria by the plasmid. Thereby when the bacteria are carrying a high-copy plasmid like pSB1C3-K343004 it is to expect that the bacteria will quickly shed the plasmid when no longer exposed to a selection pressure.
It is likely to believe that pSB3C5-K343004, since being a low-copy plasmid, will not excert as much strain on the bacteria, and might therefore be stable for more genrations than pSB1C3-K343004. Therefore a stability assay of this plasmid might be of interest.
It is likely to believe that pSB3C5-K343004, since being a low-copy plasmid, will not excert as much strain on the bacteria, and might therefore be stable for more genrations than pSB1C3-K343004. Therefore a stability assay of this plasmid might be of interest.

Revision as of 14:56, 27 October 2010