Team:Chiba/jk/γ/Trl

実験ログ 出力班

purpose
To find the earliest output gene after induction

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)
 * 46-well plate(deep well)
 * 96-well plate(deep well)
 * Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
 * Beckman Allegratm X-12R Centrifuga(Beckman Coulter)

Method
Strain:XL10G KanR


 * Liquid medium experiment
 * 1) Pre-culture
 * 2) Picked and cultured the following plate 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) Culture
 * 7) Dilute pre-cultures and add to new LB medium.
 * 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 about 6 h
 * 11) Wash
 * 12) Transfer 10mL each of the culture to 50mL centrifuge tubes.
 * 13) Centrifuged for 6min at 3600rpm,20°C and discarded the supernatant.
 * 14) Add physiological saline and resuspention
 * 15) Repeated wash twice.
 * 16) Add M9 minimal medium.
 * Mix
 * 1) Dispens each culture into 48-well deep well or 96-well deep well
 * 2) Add IPTG fainal conc. 0.2 mM (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
 * 3) Add AHL fainal conc. 100nM (BBa_T9002, BBa_K084003)
 * 4) Measure fluorescence intensity every 1 h.

Result

 * 28,August,2008
 * 5,September,2008
 * 10,September,2008
 * 14,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.