Team:Virginia/Modeling

There are two primary relationships that are necessary to model and obtain experimental results for in order to obtain a high yield of fully functioning minicells. See Figure 1 for a visual representation of the statements below.

1. The amount of IPTG needed to cause a desired amount of overexpression of FtsZ protein. The change in the concentration in the IPTG will cause a resultant change in the overall concentration of FtsZ present.
2. The FtsZ protein amounts per cell needed for circular minicell formation. The concentration of FtsZ protein in the parent cells will determine how often a circular minicell will form from its parent cells division.




IPTG

The amount of the promoter inducer added (in this case IPTG) will directly affect the production rate of the FtsZ and GFP. By default, the IPTG inducible promoter is switched off and FtsZ and GFP production should not occur without IPTG present (this is assuming no leakiness of the promoter). Additionally, the level of IPTG in the cells should remain relatively constant after a short period of time as IPTG will not be degraded in the cell and it is a small enough particle to be able to diffuse into the cell at a decent rate. The IPTG inducible promoter actually relies on lac operon infrastructure, which can be seen in Figure 2. This means that this plasmid will only be able to achieve controllable minicell formation in cells that have the lac operon and have the FtsZ Z ring formation complex as their mode of division.

Controlling the IPTG concentration of the growth medium is simple enough as its concentration will not change with time. Hence, by adding the appropriate number of moles of IPTG, the concentration can be controlled. This IPTG is added after allowing the cells to reach a reasonable optical density (the standard is approximately 0.1 A).





FtsZ

Spherical minicell formation will only occur if the FtsZ concentration/ the FtsZ amount per cell falls within a certain range. Below a lower boundary threshold value, there will not be minicell formation of any sort and ordinary cell division will progress normally. Alternatively, if above an upper boundary threshold value, minicells will form, but other irregular, long, tubular rod cells will form that do not behave like the desired spherical minicells form as well. The further above this threshold boundary value, the greater the prevalence of these rod cells. Hence, it is of paramount importance to properly control the FtsZ concentration/ amount per cell. This is achieved using a sole additional copy of FtsZ in the plasmid construct. Using this in conjunction with the IPTG promoter allows for quantifiable control of the FtsZ production so that the desired minicells can be formed (Refer to Results section for details on how FtsZ production can be quantified).





Indications of Tinkercell Modeling

By retrieving and/or estimating some of the initial values and parameters for the relative concentrations of the involved proteins, the degradation rates of the involved proteins, ribosome binding site affinity, transcription rates, ect., projections of the protein production levels were made.




Qualitative Observations

Regardless of how much IPTG is added to the system (so long as it is greater than 0), then there will be increased GFP and FtsZ concentrations in the XL1 Blue. Because of the negligible degradation rate of GFP, it became apparent that after several cell cycles, there would likely be an accumulation of leftover GFP. Similarly, if FtsZ is overproduced to a significant degree, then there may actually be FtsZ from previous generations of cells that do not get degraded quickly enough to inhibit buildup of FtsZ protein in the later generations of parent and minicells. This meaning that the undesirable rod shaped cells are still likely to form in most of the samples given too long to grow without being purified.




Quantitative Observations

The model as well as experts in the field of minicells (Matt Giacalone) indicated that using IPTG concentrations of over 100 µM would likely lead to rods forming quickly rather than the desired spherical minicells.