Part:BBa_K1649002:Experience

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Applications of BBa_K1649002

The chimaera Cph8 is the phytochrome Cph1 fusing with a cyanobacterial photoreceptor EnvZ which can engineer a red light-regulated transcription system in E. coli, with the phycocyanobilin-biosynthesis genes (ho1 and pcyA). The promoter OmpC is repressed by the function of red light and activated by dark .As the transcription from the red light sensor is repressed by the function of red light and activated by dark, we use a not gate, repressor TetR to ensure the system is activated by red light.GFP can be used to test whether the circuit works.

Measurement

Design
This part is a red light sensor combined with a tetR not logical gate followed by a GFP reporter. GFP can be used to evaluate the efficiency of red light sensor when this part is regulated by different promoters by measuring the florescence. In our experiment, the promoter is T7 promoter which is originally on pET28a and can be regulated by IPTG. In the preliminary experiment, we have searched out the suitable concentration of TPTG which are 0.6 mM and 0.8 mM. The sensor is inactive in dark and active under the red light.
Plasmid Construction.(gene circuit)

BBa_K1649001 was cloned into pET28a using the NotI and XbaI sites. This plasmid contains a Kanamycin resistance gene and a pBR322 origin.
Strains
BL21 strains are used to harbor red light sensor plasmid. The control is BL21 strains.
Culture
BL21 strains transformed with red light sensor plasmid and BL21 strains ( negative control) were each picked 5 colonies and grown overnight (~12 hours) at 37℃ and shaken at 200 rpm in 3 ml LB containing antibiotic Kanamycin. The overnights were then diluted 1:500 into 100 mL fresh media in flasks and shaken at 200 rpm for 2 hours at 37℃. Group 1: 5 flasks containing BL21 strains that harbor red light sensor plasmid and adding antibiotic Kanamycin and 0.6 mM IPTG. Group 2: 5 flasks containing BL21 strains that harbor red light sensor plasmid and adding antibiotic Kanamycin and 0.8 mM IPTG. Group 3: 5 flasks containing BL21 strains and adding antibiotic Kanamycin and 0.8 mM IPTG. Each strain were then taken 200μl into 2 new, identical set of 96-well plates. One plate was exposed to red LED which we call p1 and the other were wrapped in aluminum foil and kept in darkness which we call p2. All plates were maintained at 37℃ and measured OD600 and fluorescence at intervals.

Result
From the statistics, we have found that 0.6 mM IPTG has a better induction, so the dates shown below are from 0.6 mM IPTG induced experiment.

Fig.1The figure describes fluorescence level changing over time. Obviously the fluorescence in light is higher than in dark. It proves that our part does work with the presence light.

Fig.2This figure describes the bacterial growth curve (OD600 value).The figure is much similar with the theoretical curve and prove that our protocol, plate reader and bacterial work well.

User Reviews

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