IGEM:IMPERIAL/2006/project/Oscillator/cellcre

This is not nearly as simple as we had thought.

The original plan was to use a CRE containing plasmid which would have an antibiotic resistance gene such as chloramphenicol and use a similar plasmid with a marker such as LacZ that has the same resistance gene. So both cell populations are resistant to the antibiotic.

However

Cre can recombine the excised DNA back into the plasmid so the commercially available plasmids have clever mechanisms for creating a pulse of CRE recombinase upon activation eg.

"705-Cre plasmid is designed for use in Cre-mediated genomic manipulations. The plasmid has a pSC101 origin which maintains low copy and replicates at 30°C. The plasmids will not propagate and will get lost when incubated at 37°C. JS: I don't understand this. What do you mean by get lost? JC: they willl be removed form the system by some mechinism however it is not important to the system how the cre plasmid is removed  The expression of the Cre-recombinase is driven by the thermo sensitive promoter cI578 (λPR promoter). Therefore, the expression of Cre is repressed at 30°C and induced between 37-42°C. The plasmid carries a chloramphenicol resistance."

This is good in the sense that it reduces the load placed on the cells by this part, therefore should help keep the cell populations roughly constant. However it adds experimental constraints when preparing the predator cells.

To monitor the cell populations


 * Only two Plasmids can be electroporated into bacteria simultaneously
 * You cannot efficiently electroporate new plasmids into E-coli cells which already have plasmids.

Use a three antibiotic system to measure the cell populations.

Prey resistance =
 * Ensure the prey cell construct gets put into an AK plasmid (Ampicillin and kanomycin resistance)
 * The prey will be resistant to chloramphenicol during growth but the CRE plasmid will destroy itself in the working system. So the prey must be grown in Chloramhenicol and Ampicillin.

Predator Resistance =
 * Ampicillin and Tetracycline (put in plasmid PSBAT3)

The oscillator will run in ampicillin medium

You take a sample from the oscillator, dilute and plate on Tet or Kanomycin medium then count the colonies. JS: This method is good, but will not allow us to "instantaneously" measure the populations. We will need to wait overnight at least to get a measurement  This is far more accurate than a flourescence/lacZ mesurement and it is much easier to perform. any other method of mesurement does not give us any controll over the populations so knowing that one is falling "instantanioulsy" as a pose to the next day when we analyse the data dosn't help us in any way.

You can also control the amount of each population in the culture by adding small amounts of their respective antibiotic (sub lethal to the other pop) to increase their relative fitness, therefore change the proportion of the culture made up by predator/prey. JS: How much does it take to kill a cell? [registry]

This is the easiest way of measuring cell pop and it allows the populations to be controlled Does changing the concentration of antibioic actually give us a direct way of measuring the cell population? Maybe it is better to measure the cell ratios as you have below...

If we are going to mesure cell ratios this is the best way to do it. This needs to be discussed by the group.

However
 * It will take weeks to work out the concentrations of antibiotic needed to tune the culture
 * The ligations will only be relitively straightforwards and will slow down the project
 * we need to decide how important monitoring cell population really is As i am not convinced that the relitive fitnesses of the predator and prey will be that diferent JS: Please can you clarify what you mean by fitness? You may even observe a kind of self regulation effect where when there are few predators the prey cells work hard and decreace their fitness so the pop size falls and when the predator cells are in excess the AHL is mopped up so the prey cells don't work as hard so their relitive fitness increaces and the pop of prey cells increaces. You could force this kind of effect by increacing the load the Prey Construct places on the cell then tune the ratio with antibiotics but this is an extension and we will not have time to do it JS: I do believe that it is important to monitor cell concentrations and ensure that they are what we want. The relative proportions of the two cells will affect the ratio of the concentrations of AHL to AiiA, thus changing our parameters in the pseudo-LV model that we are using.  Even if we cannot get a quantitative measurement of the concentration of the cells, I think we should strive to ensure that the ratios of the two cells remains constant.  TH: David Leak said the chemostat really amplifies any difference in fitness between cell populations

LacZ

John Chattaway