Team:Chiba/jk/γ/trs

Reporter

 * Fluorescent Protein
 * GFP
 * pGFPuv
 * BBa_T9002
 * Venus YFP
 * BBa_K084003
 * pLac-Venus YFP
 * mCherry

pLac-mCherry
 * β-gal (X-gal assay)
 * pUC19(plac-LacZα)

Equipment

 * shaking incubator
 * Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37&deg;C)

Method
Strain:XL10G KanR
 * Agar plate experiment
 * 1) Pre-culture
 * 2) Picked and cultured the following plate stocks in 2mL of LB:
 * 3) LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
 * 4) LB-Amp, (BBa_T9002, BBa_K084003)
 * 5) Cultured at 37°C for 12h.
 * 6) Spread on plate
 * 7) Spread on new plate
 * 8) LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
 * 9) LB-Amp, (BBa_T9002, BBa_K084003)
 * 10) Cultured at 37°C for 12h.
 * 11) Colony lift
 * 12) Colony lift to inducible agar plate (containing IPTG or AHL)
 * 13) LB-Amp+0.2 mM IPTG agar plate, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
 * 14) LB-Amp+100 nM AHL agar plate, (BBa_T9002, BBa_K084003)
 * 15) Incubate at 37 °C
 * 16) Check expression every 30 min.

Result

 * 29,August,2008
 * 2,September,2008
 * 10,September,2008

Discussion Discussion

The purpose of this project is to alter the time required for expression.

expression to be observable decreased, but this still takes longer than the time required when using fluorescent proteins.
 * By increasing the concentration of X-gal, the time required for

output may increase because the substrate concentration decreases with time.
 * Using β-gal, which uses X-gal as substrate, the time required for

For the above reasons, fluorescence proteins are more suited for our purposes.

Of the fluorescent proteins, GFP and YFP fluorescence was observable at earlier stages.


 * But to the naked eye, GFP could be observed more easily.

Therefore, we chose GFP as our output reporter.