gfp reporter recombinase activity (prec1-off  (Addgene inc)

Journal: bioRxiv

Article Title: Long duration environmental biosensing by recording analyte detection in DNA using recombinase memory

doi: 10.1101/2023.08.10.552812

Figure Lengend Snippet: Ridgeline plots show the distribution of fluorescence in the arabinose-memory biosensor cell populations (pAra + pRec1-OFF) when exposed to different arabinose concentrations. The x-axis shows the fluorescence intensity, while the y-axis shows the relative abundances of cells at each intensity. The relative cell abundances of each sample were independently normalized, such that peak amplitudes are not comparable across different experiments. The vertical line within each peak and the numbers below each peak indicate the median intensity values. Three controls are shown, including ON which is pRec1-ON, the OFF which is pRec1-OFF, and glucose which is the arabinose-memory biosensor in the presence of 0.5% glucose, which represses expression. The signals from controls were measured for two days plotted and are shown in pink (day -1, d-1) and blue (day 0, d0). Cells exposed to higher arabinose concentrations shift to higher fluorescence, except with the highest concentration (1 mM arabinose); this trend is thought to arise due to the burden of recombinase expression at the highest analyte concentrations. We gated all distributions to 99 percentile of the d-1, OFF state control.

Article Snippet: Vectors for arabinose memory (pAra) and the GFP reporter of recombinase activity (pRec1-OFF) were obtained from Addgene ( ).

Techniques: Fluorescence, Expressing, Concentration Assay

Journal: bioRxiv

Article Title: Long duration environmental biosensing by recording analyte detection in DNA using recombinase memory

doi: 10.1101/2023.08.10.552812

Figure Lengend Snippet: We analyzed the memory-reporter plasmid containing the flipping region to understand if plasmid mutations were responsible for memory loss. We conducted the long duration memory assay on cultures containing only reporter plasmids in both states (pRec1-OFF and pRec1-ON) without the sensor plasmid. We grew 25 ml cultures of the population from day 0 and day 8, extracted the plasmid and ran on agarose gel. We found that the ON state plasmid has a large deletion (∼1.4 kb), which causes the loss of both the fluorescence and the flipping region over the 8 days. We designate this mutated memory plasmid as the BROKEN state.

Article Snippet: Vectors for arabinose memory (pAra) and the GFP reporter of recombinase activity (pRec1-OFF) were obtained from Addgene ( ).

Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Fluorescence