Difference between revisions of "Part:BBa K4164018"

Latest revision as of 02:29, 14 October 2022

Contribution

This composite part is used to do further research on the yellow fluorescent protein (YFP), which is commonly used as the reporter of a gene circuit. We chose the Bobrick BBa_K592101 from the iGEM distribution kit and recombined it to plasmid pET-29a(+) by homologous recombination. We firstly studied the effect of different concentration of IPTG(Isopropyl-beta-D-thiogalactopyranoside) on the expression of YFP and the fluorescence intensity of bacteria. After that, we also extracted the YFP from the bacteria and identified the effect of temperature on YFP activity.

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 1). 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.

Figure1.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.

<b>Phenotypic Photos

Figure3.a.Effects of different IPTG concentrations on YFP expression b.YFP incubate crude enzyme solution at different temperatures for 30 min.

Sequence and Features