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For plasmids is so much shorter than their host cell's genome (about 1/1000 in my case), it will take only 1/1000 time for it to replicate.

With respect to cell cycle, when will that replication occur?

  • Description of the situation I'm facing:

    I have a low copy number plasmids (pSC101) in E.coli, and I'm measuring a fluorescence protein expression level. The problem is, even if the plasmid copy number is known (N), depending on when it is replicated, the number of plasmid in my cell can vary. Therefore, I cannot directly infer expression level per plasmid. Higher expression level might just result from higher number of plasmids.

    For instance, if it is replicated right after cell division, I will have 2N plasmids for the most of my cell cycle.

    If on the other hand, it gets replicated right before cell division (and partitioning machinery delivers exactly half the number to each daughter cell), I will have N plasmids per cell for the most my my cell cycle.

So, when do plasmids get replicated?

I could not find a similar question here with search. Thank you very much in advance.

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I've found a nice review that has many details on plasmid replication in general, and several papers about pSC101 in detail, and I'll try to extract the key information from these papers.

First of all your plasmid as an ori region that contains so called iteron:

In many cases, the origin of replication contains directly repeated sequences, termed iterons, which are the binding sites for the plasmid-encoded Rep proteins and which have control properties. (...)iterons have been described for several replicons like P1, F, pSC101 (...).

pSC101 plasmid has three of these iterons present, along with a protein called rep that is necessary for replication. Besides these there are sequences that provide binding sites for host proteins such as DnaA and integration host factor (IHF).

Details of these can be found in this and this paper, both provides details on replication and the function of ori region in your plasmid.

As stated in this other paper, plasmid copy number is controlled by the plasmids themselves, that may vary on host type and growth conditions but:

At steady state, the 1 number of replications per plasmid copy is exactly one per cell generation. This defines the steady state as shown in Figure 1. It should be stressed that while this is true for the population, it is not for individual plasmids-these are selected randomly for replication.

fig1

So not all plasmid copies are replicated once per cell cycle rather random ones are selected and they replicate to maintain the desired number. When your plasmid has reached its target copy number it will adjust its replication frequency to maintain this number. This is achieved by negative control loops. There are many ways to do this (repressor proteins, anti-sense RNA) or in your case iterons. These sequence along with the rep protein may inhibit replication if too much plasmid is present by a phenomenon called “steric hindrance" or “handcuffing”. This happens because the rep protein is capable to bind two iteron, simultaneously. At low copy numbers saturation of iteron sequences by rep proteins initiate replication, but at high copy number rep proteins tend to interact with each other and plasmids pair up through rep-rep interactions therefore cannot replicate. See figure below: rep handcuffing

So, long story short: plasmid replication in part depends on host factors but plasmids have means to control their copy number, and once target has been reached they maintain this number in a stable form. I know this answer does not provide solution to the when part of your question, but it provides an answer to the practical part of your problem namely: plasmid numbers are quite stable after the target number has been reached. I hope this helps

EDIT: I've found this article http://www.ncbi.nlm.nih.gov/pmc/articles/PMC210920/pdf/jbacter00181-0364.pdf that says:

In all experiments, plasmids pSG21 (oriS only), F'lac (oriS oriV),pBR322, and pSC101 were capable of replicating at all stages of the division cycle, with no detectable differences in the patterns.

So bad news for you, because according to the paper above you cannot tell when your plasmid actually replicates. Yet still as the average copy number is quite stable due to reasons described above this may not pose a big problem. Keep in mind even if you have an extra plasmid for a short time that may still not cause problem, because transcription of your riporter gene, folding, etc takes time under which previously synthesized proteins might degrade.

However this paper says the F+ plasmid replication is cell-cycle specific, you may wan to give it a try.

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  • $\begingroup$ Thank you so much for this help! Great to know the detailed mechanism of copy number control of pSC101 plasmids (iterons). Great papers, too. It will be great if I can also know when the replication happens, too. I have to estimate expression level per cell per plasmid and when I see twice as much fluorescence, I'd like to know whether expression level per plasmid actually went up or it's staying the same while there's just another copy of that plasmid. But thank you so much for this nice explanations! $\endgroup$ – Neil Apr 23 '15 at 16:02
  • $\begingroup$ @Neil - I did some editing, does this help you? $\endgroup$ – Nandor Poka Apr 23 '15 at 21:18
  • $\begingroup$ Yes, it is helping me greatly! Thank you so much. I'm trying my best to understand the new paper better to see if I should conclude the thought here or not. $\endgroup$ – Neil Apr 24 '15 at 15:37

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