Circuit Regulation

Aim and Motivation

The aim of the circuit regulation aspect of E-Mortality is to regulate the amount of telomerase produced in a cell through controlling the expression of c-Myc, which induces the upregulation of telomerase through the hTERT promoter.

However, we only want to induce so much c-Myc so that enough telomerase is produced to maintain the length of telomeres. This is because we do not want the telomeres to continuously extend, for that might lead to unlimited cell replication, nor do we want the telomeres to reduce in size, for that would induce apoptosis. Rather, we hope to regulate the levels of telomerase so that at a certain point in time, there is always the amount of telomerase necessary only to maintain telomere length.

Therefore, our circuit cannot constitutively express c-Myc, for that would mean that the hTERT promoter would constantly express TERT and increase levels of telomerase. This constitutive expression would lead to uncontrolled telomere extension.

Instead, rather, we choose to use an oscillator to express doses of c-Myc, and thus induce doses of telomerase production. To accomplish expressing these doses of c-Myc in order to maintain a constant amount of telomerase, we employ the genetic oscillator.

The Three Component Oscillator

Aim and Motivation

Initially, we chose to use the three component repressilator to express c-Myc. This oscillator consists of three different promoters and repressors, of which c-Myc could be put behind any one repressor coding region.

The goal of the 3 component oscillator is to create a way to regulate the amount of c-myc expressed. By using a repressilator mechanism to regulate the amount of c-myc expressed we can control the expression of hTERT and therefore the mount of telomerase expressed. The amount of telomerase will then determine the length of the telomeres at the end of the chromosomes.

This oscillator would express c-Myc in doses, as it does the repressor proteins, through a series of promoter inductions and repressions.

However, we were not sure if the genetic oscillator would be successful. Therefore, while building the oscillator we made sure to test each part of the oscillator. The first was the efficacy of repressor repression.

Experiments: Testing Repressor Efficacy

Limitations of Three Component Oscillator

Upon more detailed inspection of the models used to develop the oscillator, we found several assumptions that were used to idealize the construct and thus allow it to oscillate. These assumptions were:

1. Equal promoter strengths of pTet, pLac, and pcI
2. Equal repressor half lives of TetR, LacI, and cI
3. Equal dissociation constants of TetR, LacI and CI

Under all of these ideal conditions, our models of the repressilator looked as follows:

The promoter strength, repressor half-lives, and dissociation constants were all assumed to be the same in models of the three component oscillator. However, we found evidence that promoter strengths, dissociation constants, and repressor half lives for components in the oscillator are different. We thus decided to test if differences in promoter strengths, dissociation constants, and repressor half lives alter ideal oscillatory behavior. These results are shown below:

This graph shows that changing the promoter strength changed the peaks of the proteins and had little effect on derailing the oscillation. On the other hand, changing the half-lives and dissociation constants had profound effects on oscillator behavior. After making LacI half-life five times as long, the oscillations damped quickly and the system tended towards equilibrium. The oscillator did show some resistance to reducing half-lives, but overall it showed that differences in protein half-life can entirely derail the oscillations. Changing the dissociation constant to the more realistic estimates computed by the 2009 Aberdeen iGEM team had an even more profound effect; the system immediately tended towards equilibrium, as more repressors were needed to overcome the effect of dissociation.

This shows that with realistic assumptions of differing promoter strengths, repressor half lives, and repressor dissociation constants, the oscillator fails to operate. This following model shows the change when all three parameters are changed:

Any oscillations are quickly dampened and the system moves to equilibrium. This result affirmed our concerns about the oscillator and directed us to design an alternative oscillator that is more likely to oscillate in reality.

Experiments: Testing Promoter Strength

The Two Component Repressilator

Aim and Motivation

Because we realized that the three component oscillator was too idealized and had a high probability of not working in reality, we took it upon ourselves to design a new oscillatory system that would oscillate without making the idealized assumptions of equal promoter strengths, repressor half lives, and repressor dissociation constants.

Construct Design

Instead of trying to complicate our construct, we decided to focus our efforts on designing a two component repressilator system based on the repression of only one promoter, and dissociation of only one repressor.

The design we came up with is as shown below:

Construct Design

1. When the bacteria is first transformed, both constitutive promoters pLac and pTet are turned on.
   a. This increases LacI expression
2. The LacI that is expressed goes to repress both pLac promoters, which shuts down the LacI and TetR/RFP expression.
   a. This decreases LacI expression
3. After the LacI degrades from the pLac promoter, the pLac promoter is once again turned on, expressing LacI.
   a. This increases LacI expression
4. The LacI that is now expressed goes to repress the pLac promoters.
   a. This decreases LacI expression

This is the two component oscillatory cycle. The oscillator is dependent upon the degradation of the LacI repressor, as can be seen from the oscillatory mechanism. We modeled the oscillator under experimental conditions (see modeling for further details), and found that the construct still oscillated, which was not the case for the three component repressilator.

Benefits of the Two Component Oscillator

This two component oscillatory design has numerous benefits, which are not included in the three component repressilator:

1. Promoter strength differences do reduce oscillation:
   The promoter strength differences do not destabilize the oscillator, because the regulation is solely based on pLac.
2. Repressor half-life differences do not reduce oscillation:
   The two component oscillator is more easily tuned for oscillations because only one repressor is involved in the regulation. This makes it easier to modify oscillator behavior through altering repressor half-life through half-life reducing tags such as ssrA.
3. Repressor dissociation differences do not reduce oscillation:
   The two component oscillator handles the experimental dissociation constants for the repressors better than the three component oscillator did.
4. Synchronization of oscillatory peaks can be achieved:
   Sychronization of the oscillations in groups of cells is not a problem, because it can be tuned using Isopropyl-β-D-Thiogalactopyranoside (IPTG).

IPTG is a chemical compound that allosterically inhibits the LacI repressor from binding to the pLac promoter. By using IPTG to disable LacI in a group of cells, all the oscillatory systems will shift to maximize pLac efficiency. When IPTG naturally degrades, the LacI will resume expression from the same oscillatory phase for all the cells in a group, thereby tuning the oscillations.

In the normal three component repressilator, because the oscillatory phases of different cells are different, noise will be created, making the oscillator phases untuned. This noise would look like the following graph:

However, we are able to synchronize the oscillatory constructs through use of the IPTG chemical compound, which would cause the peaks to become more aligned.

Taken together, using the same modeling equations as used for the three component repressilator, yet not assuming ideal conditions, our two component system is still seen to oscillate, as demonstrated by the model below:

Benefits

The comparisons between the three component traditional repressilator and the two component oscillator are summarized in the table below:

Table 1. Table to show the differences between the three component and two component oscillators.

	Three component	Two component
Promoter strength assumption	Promoter strengths must be the same for ideal oscillation	Promoter strength does not affect ideal oscillation
Repressor half-life assumption	Repressor half lives must be the same for ideal oscillation	Repressor half lives do not have to be the same for ideal oscillation
Dissociation constants	Dissociation constant differences among repressors must be the same for ideal oscillation	Dissociation constant differences affect ideal oscillations much less
Synchronization	IPTG synchronization reflex is slower	IPTG synchronization reflex is quicker
c-Myc expression and tunability	Overexpresses c-Myc under virtually all conditions, so harder to tune	Easier to tune than three-component oscillator, c-Myc expression can be tuned under control

Modeling of the two component oscillator

After the oscillator settles, it is able to express the number of c-Myc proteins that are necessary to maintain telomere length. Specifically, the oscillator is able to maintain a level of 1000 c-Myc proteins, which is significantly more than the 616 minimum number of c-Myc proteins necessary to maintain telomere length. The initial spike is caused by the constitutive promoters pTet and pLac being activated when first transfected into the cell.

Therefore, our models suggest that our oscillator is capable of expressing the necessary concentrations of c-Myc.

Results from building the two component oscillator

In order to test the oscillator, we fused the proteins TetR to mRFP to use for visual testing. We submitted this newly created BioBrick as K1253100.

We then built the first half of our oscillator, which looks like this:

We then inserted the pLac+LacI to complete our oscillator design. This new part we name K125112, which we also submitted to the registry as a new oscillator design. The construct is depicted below:

The constitutively expressed LacI repressed the pLac promoters, which repressed the expression of TetR/RFP, and hence allowed the pTet promoter to express green. As expected, the colonies in which we transformed glew green, as shown below:

These prove that our repressor mechanisms were successful in changing from the green peak to the red peak based on the self-regulating mechanism of pLac+LacI.

Conclusion

Overall, the two component oscillator has a simpler design and is thus less constrained by differences of promoter strength, repressor half lives, and repressor dissociation constants. This makes the two component oscillator more realistically implementable.

We also determined that the two component oscillator is able of expressing the number of c-Myc proteins necessary to maintain telomere length.