Insulin Red Fluorescent Protein Proof of Principle

TAT signal sequence

RFP with Cells Media at Different Induction Levels

Preliminary results

Cultures were grown containing LacI promoter attached to a twin arginine tag (TAT) signal sequence and fused to a red fluorescent protein (E1010). Samples were taken at various time points and examined for fluorescence when excited at 586nm with an expected emission at 607nm. We used a fluorescence spectrophotometer to measure the red fluorescent protein present in the supernatant after spinning the cells to a pellet. We used a constutively expressed construct expressing Red fluorescent protein( J23100 in J61002)

Fluorescence was measured one hour after induction, no difference could be seen between the test and control constructs in the supernatant .

Fluorescence was measured two hours after induction, no difference could be seen between the test and control constructs in the supernatant .

Fluorescence was measured three hours after induction, no difference could be seen between the test and control constructs in the supernatant .

Fluorescence was measured four hours after induction, no difference could be seen between the test and control constructs in the supernatant .

Glucose Concentration Assay

Preliminary Results

Cultures were grown containing constitutively expressed promoter J23100 attached to an MlC binding sight sequence and a red fluorescent protein. Samples were inoculated in varying concentrations of Glucose (0=0 mg/dl, 20= 12.5mg/dl, 40=25mg/dl, 80=50mg/dl, 160=100mg/dl, 320=200mg/dl, 1600=1000mg/dl). Samples were taken after 1.5 hour time points post inoculation and examined for fluorescents when excited at 586nm with an expected emission at 607nm. We used a fluorescents spectrophotometer to measure the red fluorescent protein present in cell lysate after spinning the cells to a pellet and re-suspending in 8M urea.

Fluorescence was measured three hours after inoculation, no RFP emission differences between cells grown in the different glucose concentrations were observed.

Fluorescence was measured four and a half hours after inoculation, no RFP emission differences between cells grown in the different glucose concentrations were observed.

Fluorescence was measured six hours after innoculation, no RFP emission differences between cells grown in the different glucose concentrations were observed.