Part:BBa_K3484006

sfGFP + ASV tag

The superfolder green fluorescent protein (sfGFP) is a variant of the common green fluorescent protein, that folds faster, thus generating fluorescent in less time.

Figure 1. Experimental data extracted from the Plate-reader. Each curve is a mean of 4 different wells to reduce noise.

Model Characterization

In order to quantify how the ASV tag affected the degradation rate of the sfGFP, a simple mathematical model was developed. The following model is exactly the same for the two scenarios, and consists of a constitutive expression minus a degradation rate (Equation.1). As both systems are constitutively regulated by the same promoter, the PJ23100 (BBa_J23100), the production rate will be the same in the two systems. In addition, the equations representing the steady-state (dy/dx=0) of the system were calculated (Equation.2).

Equation 1. Ordinary Differential equations for the sfGFP cell with (B.) and without tag (A.).

Equation 2. Steady-State equations for the sfGFP cell with (B.) and without tag (A.).

With the Steady-State equations we are able to look for the relationship between the two degradation rates (δ) as the production rate is the same(Equation.3).

Equation 3. Relationship between the two degradation rates (δ) with and without tag (The last point of the experimental data was taken as the steady-states).

In the relation between the degradation rates (Equation 3.) we can clearly see that the ASV tag has an impact on the degradation rate. Mathematically it has been proved that the degradation rate is increased, approximately, 25% when the ASV tag is added on the sfGFP gene sequence. All in all, it has been demonstrated that the addition of the ASV tag increases the degradation rate. Therefore, the introduction of an ASV tag is a good approach to reduce the recovery system time without losing effectiveness of measurement, which is interesting for any kind of biosensor or feedback loop application.

Characterization experiments

For the characterization a Plate-Reader analysis was made. All the information on the experimental conditions and parameters used are described on the table below (Tab.9).

Table 1. Plate-Reader Parameters for the characterization of the effects of ASV tag in sfGFP
Parameters	Value
Plate-Reader model	Synergy HTX
Plate type	Thermo Fischer 96-well microplates black-walled clear bottom
Cell medium	LB
Time	24 hours
Shake	Linear: Continuous, Frequency: 567 rpm (3mm)
Temperature	37C
Gain	50
Optical Density (OD) measurement (absorbance)	660nm
GFP excitation wavelength	485nm
GFP emission wavelength	528nm
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