Reporter
sfGFP+ASV

Part:BBa_K3484006

Designed by: Tomas Berjaga, Quim Marti & Jaume Puig   Group: iGEM20_UPF_Barcelona   (2020-10-25)
Revision as of 23:10, 25 October 2020 by Tomasberjaga (Talk | contribs)


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).

Characterization experiments

For the characterization of all the cell types described in this page Plate-Reader analysis were made. Although GFP emission was measured to check the sensor activity in all the experiments, RFP emission was only used when the experiment required 2 cells per well. Measuring the RFP emission allowed us to know the optical density of the 2 cells separately. 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

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 25
    Illegal AgeI site found at 148
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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