Copy number control

How do we control plasmid replication

We were disappointed with the results of testing red gradients under central IPTG control. While the results demonstrated the functionality of control, even when completely repressed the colonies would slowly turn pink over the course of a couple of days.
[xxx describe concept. include figure]

Method of measuring repressor-controlled plasmid loss using a cell viability assay.

Bacterial cultures were grown at 37OC in LB broth (xxx) overnight under selective conditions that favoured plasmid maintenance [chloramphenicol (xxx/mL) and either IPTG (xxx) or ATC (xxx)]. As a starting point, the number of viable cells in each culture was determined by spotting 5uL of 10-fold serial dilutions ranging from undiluted to 10-9 (xxx spots/culture) onto LB-agar plates containing Chloramphenicol and either IPTG or ATC at the concentrations cited above. Cell count/mL of culture was determined by counting (or estimating) the number of colonies at the highest resolvable dilution and multiplying by the dilution factor.
Fresh cultures were then made by inoculating one uL of the original cultures into 5 mLs of LB broth under conditions that favoured plasmid loss (no antibiotic or inducer) and grown as above. The number of viable cells remaining under non-selective conditions were determined as described above.

Mathod making Inducer gradient plates

Making the plates. LB agar plates were made using 25 mL of LB agar. A well in each plate was made using two cylindrical neodymium magnets (dimensions xxx) per well that sandwich the lid at the preferred location, as illustrated below. When solidified the magnet is removed by gently lifting off the lid.
Making the gradients. 40uL of either 100x IPTG (10mM) or 100x ATC (100mM) are added to each well and allowed to diffuse for 8 hours at room temperature prior to plating.
Plating bacterial lawn. Overnight cultures are diluted to an approximate cell concentration of between 0.5-1.0x105 cells/ml. 1-2x104 cells in 200 uL are plated by swirling 6 ball-bearings (dimensions xxx) on the plate surface until completely wetted. The plates are then dried under a tissue culture cabinet and incubated at 37OC overnight.
Important! Cell densities that exceed those specified above produce thin transparent lawns with poor colour development.

Aplication in project