Purpose & Design
For the contribution part of our project, we decided to do further research on the Yellow Florescence Protein (YFP), which is commonly used as a reporter in the system. We chose BBa_K592101 from the iGEM distribution kit and used pET-29a(+) as the vector, which is capable of expressing LacI to conduct BBa_K4164018. Then, we transformed it into Escherichia coli DH5α to grow and E. coli BL21 to express, then studied the effects of different concentrations of IPTG on the expression of YFP and the fluorescence intensity of bacteria. After that, we extracted YFP from the bacteria and identified the effects of temperature on YFP activity.
Figure 1. Contribution of YFP expression device
Experiment
BBa_K592101 was obtained from the distribution kit and transformed into E. coli DH5α. Then the bacteria were smeared on the LB plate, and cultured overnight at 37℃. The next day we picked 4 colonies and incubated them in 5mL LB medium for about 12 hours.
This part was expressed in plasmid pSB1C3. So the LB plate and medium we used were supplemented with chloramphenicol (50μg/mL). Since the original plasmid did not encompass components such as promoters, RBS and terminators, we built the purified sequence into the pET-29a(+)plasmid. The final plasmid was mainly composed of T7 promoter, an operon sequence of lac which is sensitive to IPTG, YFP coding sequence and T7 terminator.
1. The effects of different concentrations of IPTG on E. coli BL21 containing BBa_K4164018
The plasmids were extracted and transformed into E. coli BL21 and cultured overnight in 5 mL LB medium.
The overnight culture of E. coli BL21 was diluted into fresh 50mL LB medium and incubated at 37℃ to a concentration of 0.6 optical density at 600nm [OD600], then they were separated into 6 groups, corresponding to the 6 different IPTG concentration gradients 0mM, 0.1mM, 0.5mM, 1mM, 5mM and 10mM.
The bacteria medium was incubated with a shaking rate at 200rpm and sampled every 1 or 2 hours and measured OD600 as well as fluorescence data in a 96-well microplate, with LB media served as control.
2. The effects of temperature on YFP activity
After taking out 50mL cell culture, we centrifuged it at 8000 rpm for 10min, and discarded the supernatant. Then, we added 10mL Tris-HCl(trishydroxymethylaminomethane-hydrochioric acid) buffer to the precipitate and made it re-suspended. The cells were then crushed by ultrasonic for 10min. The supernatant was obtained as the YFP crude protein solution.
Subsequently, we incubated the YFP solution at 4℃, 16℃, 37℃, 42℃ and 55℃ for 30min. Then we measured their fluorescence intensity in a microplate reader（excitation 484nm; emission 534nm). The crude protein solution with ddH2O of the same volume was prepared simultaneously to serve as the control.
Results
1. The effects of different concentrations of IPTG on E. coli BL21 containing BBa_K4164018
We compared the inducing effects of IPTG on YFP through different concentrations of IPTG. According to the result data, no significant differences in fluorescence intensity were observed by using different concentrations of IPTG (Figure 2). What's more, we could deduce that the leakage expression was very low and the bacteria was sensitive to the IPTG induction. For the first four hours, the fluorescent intensity did not differ much under IPTG induction, among which 0.5mM showed the best. While after 4 hours of incubation, the fluorescence intensity rose sharply, reaching its highest value at the sixth hour, and remained stable with slight fluctuations ever since.
Figure 2. The effects of different concentrations of IPTG on E. coli BL21（DE3） containing BBa_K4164018
2. The effects of temperature on the activity of YFP
It is derived from the literature that the α-helix at the closed end of the structure of green fluorescent protein extends inward to the mid-axis to become a scaffold for the chromophore. The chromophore is at the core of the entire structure and is protected by the peripheral β-folded sheet layer. Such a structure ensures a high stability of the whole structure and greatly improves the resistance of the core chromophore to heat, acid, alkali and denaturant factors. Therefore, we speculated that the effect of temperature on the yellow fluorescent protein was mainly on the protein, while there was no significant effect on the chromophore. From the data, we can see that there were some differences in the activity of YFP at different temperatures for E. coli BL21 (Figure 3). The results indicated that YFP activity was higher in low temperatures (eg. 4℃, 16℃ and 37℃) and lower in higher degrees. According to the curve, the value of fluorescence intensity of YFP firstly witnessed a slow drop from 4℃ to 16℃. Then it climbed steadily and peaked at a point somewhere before 37℃, which met our expectation. This confirmed our suspicion.
Figure 3. The effects of temperature on YFP activity.
Phenotypic Photos
Figure 4. a.Effects of different IPTG concentrations on YFP expression b.YFP incubate crude enzyme solution at different temperatures for 30 min
Reference
Daubner, S. Colette, et al. "Yellow light emission of Vibrio fischeri strain Y-1: purification and characterization of the energy-accepting yellow fluorescent protein." Proceedings of the National Academy of Sciences 84.24 (1987): 8912-8916.
Zuqiang Liu, Min Hu, Yipeng Qi. "Studies on the Sructure, Mechanism of Green Fluorescent Protein and Its Application."Journal of Wuhan University(Natural Science Edition),2000,46(2):211-214.
