The improvement of parts on the iGEM registry is an important task to be fulfilled by the teams. In this page you will find how we characterized the effect of the ASV degradation tag on the super-fold green fluorescent protein (sfGFP).
Improvement of an Existing Part
As a mechanism to sense the concentration of lactone in our Proof of Concept, we used a variant of the Green Fluorescent Protein (GFP), the super-folder GFP (sfGFP). As explained in the Proof of Concept section, sfGFP was used as it produces faster responses, due to a faster folding. Faster responses are a key aspect when it comes to our system, as the feedback of the PID controller needs discrete data in a short period of time to be able to regulate. The mentioned sfGFP is already an existing part in the iGEM registry (BBa_I746916).
However, our system needs to be able to sense long periods of time and, to do this, the protein degradation rate must be increased. This phenomena allows us to have a faster recovery of the system, and therefore, to have a more rigorous and continuous control of the protein (Eq.1). But how can the degradation of GFP affect the time to sense?
By increasing the degradation rate we are decreasing the time that our cells take to arrive to the GFP steady state that corresponds to the AHL concentration present in the media (Eq.2). If we increase the degradation this final concentration of GFP will be lower, thus reducing the time to arrive to it. Furthermore, if the AHL concentration decreases, having a stronger degradation will reduce the time that the cell takes to arrive to the new, lower GFP steady state. By reducing the time that our cells need to sense the AHL concentration we can increase the number of times that we can sense in the same amount of time, thus producing a more rigorous control.
In the context of our AHL sensor cell, the best way to increase the protein degradation rate is to add a degradation tag to the sequence of the sfGFP . In our context, the ASV tag was added into the original sfGFP sequence. This addition can be seen as an improvement of an existing part, and its characterization is studied below.
Adding the degradation tag
To add the degradation tag to the sfGFP sequence, first we studied which tag would work better for our case. We decided to use the ASV degradation tag, strongly recommended by one of our advisors, Carlos Toscano, as it will ensure a slight degradation rate increase that still allows us to detect different concentrations of sfGFP . This increase on the degradation of the sfGFP was mathematically demonstrated in the Modeling page.
Once the tag was added to the sfGFP sequence, we combined a constitutive promoter (BBa_J23100), and the RBS32 (BBa_B0032) followed by the sfGFP (BBa_I746916) with and without degradation tag, to see how the tag affected the final fluorescence.
We must consider that in our sensor cell a insertion PCR was done to introduce the ASV tag, so that the sfGFP could contain this sequence. All this process is widely explained at the Proof of Concept page.
CHARACTERIZATION OF THE sfGFP + ASV TAG
To characterize how the ASV tag affects the protein degradation rate we performed a Plate-Reader analysis. Afterwards, with the results obtained, we studied the fluorescence produced by the sfGFP through time. The experimental data can be seen in the figure below (Fig.1), with each curve being the mean fluorescence of 4 different wells.
All the information on the experimental conditions and parameters used are described on the table below (Tab.1).
|Experimental parameters and conditions||Description|
|Plate-Reader model||Synergy HTX|
|Plate type||Thermo Fisher 96-well microplates black-walled clear bottom|
|Shake||Liner: Continuous Frequency: 567 rpm (3 mm)|
|Optical Density (OD) measurement (absorbance)||660nm|
|GFP excitation wavelength||485nm|
|GFP emission wavelength||528nm|
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 (Eq.3). This is widely explained at the Modeling page. In addition, the equations representing the steady-state (dy/dx=0) of the system were calculated (Eq.4).
With the Steady-State equations we are able to look for the relationship between the two degradation rates (δ) (Eq.5).
The ASV implications in the degradation rate of sfGFP
The results above explicitly let us know that the ASV tag produces an increase of the sfGFP degradation rate. The final states (Eq.4) and the relation of the degradation rates (Eq.5) show that the ASV tag produces a 25% increase on the degradation rate of the sfGFP, thus allowing a faster recovery for our system without compromising the accuracy of the measurements. Having chosen the ASV degradation tag allows us to be in a sweet spot where we have reduced the time to sense, but the tag has not increased the degradation of the sfGFP enough so that the fluorescence is highly compromised. .
All in all, the introduction of an ASV tag is a good approach to reduce the system's recovery time without losing effectiveness of measurement, which is interesting for any kind of biosensor or feedback loop application.
 Andersen, J.,Sternberg, C., Poulsen, L. K., Bjørn, S. P., Givskov, M. New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria. Applied and environmental microbiology, June 1998, p. 2240–2246