<center><figcaption><div style='padding-left: 20%;padding-right:20%; padding-top: 15px; color: #808080; font-size: 13px;'>Figure 3: Measurements of the fluorescence (A) or the steady state normalized fluorescence (B) over time of BBa_K2333432 (pTet mScarlet-I pdt E), induced at 50ng/mL ATC with and without mf-Lon, and readjusted at 85ng/mL ATC with mf-Lon. Each data point represents the geometric mean of three biological replicates, with at least 10,000 cells collected for each replicate. Shaded region represents +/- geometric standard deviation. Reporter constructs were used on pSB1C3 while a pSB3K3 version of BBa_K2333434 (pLac mf-Lon) was used.
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<center><figcaption><div style='padding-left: 20%;padding-right:20%; padding-top: 15px; color: #808080; font-size: 14px;'>Figure 3: Measurements of the fluorescence (A) or the steady state normalized fluorescence (B) over time of BBa_K2333432 (pTet mScarlet-I pdt E), induced at 50ng/mL ATC with and without mf-Lon, and readjusted at 85ng/mL ATC with mf-Lon. Each data point represents the geometric mean of three biological replicates, with at least 10,000 cells collected for each replicate. Shaded region represents +/- geometric standard deviation. Reporter constructs were used on pSB1C3 while a pSB3K3 version of BBa_K2333434 (pLac mf-Lon) was used.
An important dynamical property of this circuit is the pulse sharpness. There are two ways to tune this property: (1) By increasing the strength of Y -| Z, you make the damping to the lower steady state occur faster, which means the pulse is narrower. (2) By increasing the speed of X->Z, you make the rise-time to the peak faster, which makes the pulse taller. Both properties lead to sharper pulses. We realized that we would be able to construct a minimal IFFL circuit by using Lon’s proteolytic degradation of a tagged protein as the inhibition step in the circuit.
An important dynamical property of this circuit is the pulse sharpness. There are two ways to tune this property: (1) By increasing the strength of Y -| Z, you make the damping to the lower steady state occur faster, which means the pulse is narrower. (2) By increasing the speed of X->Z, you make the rise-time to the peak faster, which makes the pulse taller. Both properties lead to sharper pulses. We realized that we would be able to construct a minimal IFFL circuit by using Lon’s proteolytic degradation of a tagged protein as the inhibition step in the circuit.
<figcaption><div style='padding-left: 20%;padding-right:20%; padding-top: 15px; color: #808080; font-size: 13px;'>Figure 7: Time course measurement of pTet mScarlet-I pdtA induction with Lon already at steady state or induced at the same time. Geometric mean of three biological replicates, shaded region represents one geometric standard deviation above and below the mean, at least 10,000 cells collected per time point.
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<figcaption><div style='padding-left: 20%;padding-right:20%; padding-top: 15px; color: #808080; font-size: 14px;'>Figure 7: Time course measurement of pTet mScarlet-I pdtA induction with Lon already at steady state or induced at the same time. Geometric mean of three biological replicates, shaded region represents one geometric standard deviation above and below the mean, at least 10,000 cells collected per time point.
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Revision as of 21:24, 1 November 2017
Degradation Rates
When Cameron and Collins demonstrated the functionality of protein degradation tags (pdt) and the Mesoplasma florum Lon (mf-Lon) protease in E. coli, they did their work exclusively using genomically integrated constructs. Since the majority of iGEM teams work mainly with plasmid constructs, we first wanted to confirm and characterize the parts using iGEM backbones. To do this we assembled constitutive and ATC inducible constructs carrying the red fluorescent protein mScarlet-I, tagged with each of our six different pdts, or left untagged as a control. Further, to ensure that our project will work with a variety of different proteins, we made identical constructs encoding for superfolder GFP (sfGFP) and performed preliminary characterization (Figure 2).
Speed Control
Once we confirmed that degradation was working reliably, and that we did in fact have a variety of different strength tags, we then tested whether we had control over gene expression speed. Using the ATC inducible mScarlet-I constructs from the previous section, we confirmed that we could change the gene expression speed of our constructs (Figure 3a). Further, we noted that if you plot gene expression speed against degradation rate, then as we expect, the shape of the curve resembles that of a constant/degradation (Figure 3b). Together, this data represents the first experimental confirmation of the relationship between gene expression speed and degradation rate.
While we have demonstrated a change in gene expression speed, recall that the steady state value for protein concentration is given as the production rate divided by the degradation rate. This means that as we increase the speed of gene expression, we are also decreasing the steady-state value. While some applications of genetic circuits may only be concerned with a gene’s expression as an on or off signal, we wanted our system to affect speed while maintaining the original steady state protein concentration.
According to our model, gene expression speed is only regulated by degradation. This implies that it should be possible to readjust our steady-state value back up to its original expression level by manipulating protein production rate, without affecting the associated speed change. Using pdt E as an example, we measured the time to steady state with and without mf-Lon at a given ATC induction level. We then showed that by increasing the ATC concentration (increasing production rate), we can return the steady state of the with-protease condition to that of the without-protease condition while maintaining the same speed change, exactly as our model predicts.
Figure 3: Measurements of the fluorescence (A) or the steady state normalized fluorescence (B) over time of BBa_K2333432 (pTet mScarlet-I pdt E), induced at 50ng/mL ATC with and without mf-Lon, and readjusted at 85ng/mL ATC with mf-Lon. Each data point represents the geometric mean of three biological replicates, with at least 10,000 cells collected for each replicate. Shaded region represents +/- geometric standard deviation. Reporter constructs were used on pSB1C3 while a pSB3K3 version of BBa_K2333434 (pLac mf-Lon) was used.
Enabling Future iGEM Teams
Once we felt that we could understand and control gene expression speed, we next wanted to make our system more accessible to future iGEM teams. While our system is inherently easy to clone and implement, as it only consists of only a 27 amino acid residue pdt and the associated mf-Lon protease, we wanted to make it even easier to implement. With this in mind, we created a suite of ready-to-clone pdt constructs and added them to the registry. Each part contains one of our six different strength E. coli-optimized protein degradation tags with a double stop codon and a double terminator. Combining all these parts together into one construct prevents extra cloning steps, saving time, money and aggravation. In addition to the functional elements above, each construct also contains two BsaI restriction sites for Golden Gate Assembly, two Universal Nucleotide Sequences for Gibson Assembly, as well as a number of well-tested primer sequences that can be used for any other type of cloning. We also made it easy to swap and design large libraries of constructs with different speeds, by making sure that the only difference between each ready-cloning construct was a small unique region in the pdt. That means there is no need to switch primers to use a different strength pdt. Alongside our well-characterized construct Bba_K2333434 (pLac mf-Lon), these ready-to-clone parts should make it cheap and easy for future teams to test their constructs with a wide variety of different gene expression speeds, either by changing the pdt or the concentration of mf-Lon.
After demonstrating the functionality of our system using basic reporter constructs, we wanted to exhibit an example of a practical application for speed control. To that end, we collaborated with the University of Maryland iGEM team by creating and characterizing the speed of pdt-tagged versions of their copper detecting circuit. We then sent the tagged constructs back to them for future use. Though we were unable to perform the full range of characterization that we wanted due to time concerns, we found that we were able to achieve an increase in speed similar to that of our simple reporter circuits. This serves as a practical proof of concept, as we were successfully able to show that our ready-cloning parts could be used to increase the speed of an arbitrary genetic circuit.