Team:SDU-Denmark/project-p

From 2010.igem.org

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(HPLC determination of beta-carotene and retinal production)
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To dertermine the stability of our pSB1C3-K343006 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-K343006 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 generations.<br><br>
To dertermine the stability of our pSB1C3-K343006 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-K343006 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 generations.<br><br>
[[Image:Team-SDU- NinaB stab2.png|400px|center]]<br>
[[Image:Team-SDU- NinaB stab2.png|400px|center]]<br>
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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>
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-K343006 it is plausible 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-K343006 it is plausible that the bacteria will quickly shed the plasmid when no longer exposed to a selection pressure.

Revision as of 17:35, 26 October 2010