Robustness Assessment of Biomolecular Circuit with Negative Feedback

Thesis submitted by
Samarth Mohan
2016EE30516
Aman Singh
2016EE30515

Under the guidance of

Prof. Shaunak Sen

In partial fulfilment of the requirements

for the award of the degree of

Bachelor of Technology

Department of Electrical Engineering

INDIAN INSTITUTE OF TECHNOLOGY DELHI
November 2019

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 THESIS CERTIFICATE

This is to certify that the thesis titled Robustness Assessment of

Biomolecular Circuit with Negative Feedback, submitted by Aman Singh
and Samarth Mohan, to the Indian Institute of Technology, Delhi, for the
award of the degree of Bachelor of Technology, is a bonafide record of the
research work done by him under our supervision. The contents of this thesis, in
full or in parts, have not been submitted to any other Institute or University for
the award of any degree or diploma.

Prof.
Shaunak Sen
Dept. of Electrical Engineering Place: New Delhi
IIT Delhi

Date: 22nd November 2019

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 ACKNOWLEDGEMENTS

We have taken efforts in this project. However, it would not have been
possible without the kind support and help of many individuals. Firstly I would
like to express our sincere gratitude towards my supervisor, Professor Shaunak
Sen, for his continuous support throughout the project, for his guidance,
patience, motivation and enthusiasm. I couldn’t have imagined a better
supervisor and mentor for my project.

We would like to express our special gratitude to Abhilash Patel Sir (PhD
Scholar), for helping us with the experimental procedure and guiding us along
the whole project duration. We would also like to thank Krishna Kumar Golla
Sir (PhD) for his valuable contribution that we were able to smoothly conduct
the experiment and obtain proper data.

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 Abstract
The behaviour of bio-molecular circuits varies with the variation in
temperature because the rate parameter of these reactions depends on
temperature. Robustness is an important requirement for the design of
biomolecular circuits. We can improve the temperature robustness by the
cancellation of parametric dependence(rate parameters with different effects on
the reaction one affect positive and another negative) due to temperature in
synthetic oscillator but in other conditions, on another circuit, we can’t
determine the effect of temperature change. With the use of the mathematical
model and doing various experiments we can estimate the temperature
robustness of bio-molecular feedback system due to negative feedback. We can
see that the output of circuit varies with the temperature, and also we find that if
we add cancellation of parametric temperature dependance, these parameters
regimes can also increase the temperature robustness for the negative feedback
loop. We discuss these parameter regimes in the context of measured data.
These results should help in designing the temperature robust bio-molecular
circuits.

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Table Of Contents

Contents Page No.

Acknowledgement 3
Abstract 4
Table of Contents 5
List of Figures 6
Chapter 1: Introduction 7
Chapter 1.1: General 7
Chapter 1.2: Biomolecular Circuits used in experiment 8
Chapter 2: Experimental Procedure 10
Chapter 2.1: Experimental Procedure 10
Chapter 3: Observations and Results 11
Chapter 3.1: Observations 11
Chapter 3.2: Results 15
Chapter 4: Summary 16
Chapter 4.1: Summary 16
References 17

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List of Figures

Fig.No. Contents Page No.

1 Response of biomolecular circuit with temperature 7
2 Pconstitutive-TetRPtetGFP 8
3 Effect of aTc on circuit 8
4 PtetTetRGFP 9
5 Effect of aTc on the output 9
6 Description of steps in our experiment 10
7 Open Loop with 0ng aTc 11
8 Open Loop with 25ng aTc 11
9 Open Loop with 50ng aTc 12
10 Open Loop with 100ng aTc 12
11 Closed Loop circuit with 0ng aTc 13
12 Closed Loop circuit with 25ng aTc 13
13 Closed Loop circuit with 50ng aTc 14
14 Closed Loop circuit with 100ng aTc 14

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 Chapter 1
Introduction
1.1 General
We want to design our biomolecular circuits such that they behave
robustly in dissimilar environmental conditions and Temperature is one of the
environment variables. In this experiment, we will look at the effect of
Temperature on the performance of our biomolecular circuit. One of the
standard ways to implement temperature robustness is, to design and configure
parameters that are dependent on temperature, such that their combined effect
gets cancelled.
Since, the biomolecular circuits, which contains DNA, RNA, and proteins
also have temperature-dependent parameters. Therefore, we look forward to
making our biomolecular circuits robust to temperature changes.

Figure 1: The response of biomolecular circuit changes with temperature. Here,

arrows indicate the interaction between the biomolecules. The left image is the
cold temperature response and right one indicates the high-temperature
response[2].

Temperature robustness of a biomolecular circuit is an important problem

in both the natural and synthetic contexts (Fig. 1). For example, in a study[3],
where the effect of temperature on bacterial chemotaxis was observed.
And in some other studies also [4,5], it was found that the common principle
behind robustness towards temperature changes is to have circuit parameters
such that the combined effect of temperature dependence gets cancelled out.

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 In a study [6,7], it was found out, primarily computationally, that there
can be certain systems of parameters, which can also result to provide
robustness to temperature.

There are three remarkable points relating to temperature robustness in

biomolecular circuits.
1. The similarity shares with the engineering contexts, in achieving the
temperature robustness, by using suitable parameters, such that they
cancel the overall temperature dependence.
2. Biomolecular circuit outputs can be easily measured as compared to
biomolecular circuit parameters. Therefore, the problem of designing gets
reduced to just finding the right mutant, if it exists.
3. And the possibility that some system of parameters can cancel overall
temperature effect, and thus facilitating the temperature robustness.

1.2 Biomolecular Circuits used in the experiment

1. Open Loop Circuit:

Figure 2: Pconstitutive-TetRPtetGFP
Pconstituive-TetRPtetGFP is the name of our Open Loop Circuit.
aTc binds up with TetR to increase the production of GFP.

Figure 3: Effect of aTc on circuit.

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 2. Closed Loop Circuit

Figure 4: PtetTetRGFP

PtetTetRGFP is the name of our Closed-Loop Circuit and aTC is acting as

negative feedback.
In this biomolecular circuit, TetR acts as a repressor, to its own promoter
Ptet. Here, TetR represses Ptet resulting in a negative feedback loop. TetR
protein is further fused with Green Fluorescent Protein (GFP). The strength of
negative feedback can be tuned with the help of inducer anhydrotetracycline
(aTc) through its inhibitory effect on the transcriptional activity of TetR.

Figure 5: Effect of aTc on the output

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 Chapter 2
2.1 Experimental Procedure
We did the experiment in three steps which are inoculation, incubation
and characterization. So, when these three steps get completed we get our
readings for a particular temperature on that day. Same procedure we did for the
different temperature at different day.

Figure 6: Description of steps in our experiment

1. Inoculation: In this step, we grow the two circuits one is open-loop and
the other one is closed-loop in minimal media(M9CA) at two different
temperatures 29◦C and 37◦C. The culture with closed-loop circuit were
grown in the presence of kanamycin(50 ng/ml) antibiotic and the culture
with open-loop circuit were grown in the presence of kanamycin and
ampicillin antibiotic both concentration is 50 ng/ml because it contains
two individual circuits. The Antibiotic is required to use because the
contaminated bacteria will die out by using antibiotics. So that our
readings not affected by surrounding bacteria. We take the above culture
in a test-tube and put it into shaker for 16 hours.
2. Incubation: In this step, we dilute the culture in fresh media in the ratio
of 1:100 and also we add the same antibiotic which was previously used
for both cultures. We take the above culture in a test tube and put it in a
shaker for 2 hours.
3. Characterization: In this step, we add different amounts of inducer in
both circuits. The amounts we take are 0ng, 25ng, 50ng, 100ng. After
this, we take 200µl of this mixed culture into the well of a
well-plate(Perkin Elmer). Each sample was placed in triplicate. The plate
was incubated in a plate reader(Biotek Synergy H1) for 8 hours and at a
required temperature. At every 5 min, fluorescence and optical density
were measured. The measurement of the well containing only media
provided the background for these measurements. These experiments
were repeated for 29 °C and 37 °C on different days.

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 Chapter-3
3.1 Observations
1. Open Loop

Figure 7: Open Loop with 0ng aTc

Figure 8: Open Loop circuit with 25ng aTc

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Figure 9: Open Loop circuit with 50ng aTc

Figure 10: Open Loop circuit with 100ng aTc

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 2. Closed Loop

Figure 11: Closed Loop circuit with 0ng aTc

Figure 12: Closed Loop circuit with 25ng aTc

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Figure 13: Closed Loop circuit with 50ng aTc

Figure 14: Closed Loop circuit with 100ng aTc

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3.2 Results

The response of the two biomolecular circuits was measured at two

temperatures, 29°C and 37°C. And these measurements were taken on 4
different concentrations of inducer aTc.
1. We find that optical density-normalized fluorescence changes with
temperature, both in the amplitude and the response. It is also notable that
temperature is a global environmental variable that can affect other
aspects of the measurements as well, like, GFP fluorescence and its
dynamics and the aTc binding properties and its half-life.
2. We also see that for zero concentration of aTC there is almost negligible
output because as we shown earlier the closed loop circuit require the
inducer(aTC). Otherwise its promoter is not free and transcription process
stops. So, there is no protein produced in this case.But we see that in open
loop case there is some but in little amount of protein produce that
happens because for open loop case there are two separate circuits, the
output of the first circuit repress the second circuit. So, there is some
protein produced in starting after some time the production of GFP
protein stops.
3. We also see that as we increase the amount of inducer(aTC) the
production of GFP protein increase in both(open loop and closed loop)
the cases.This happens because aTC repress the effect of repressor, so as
we increase the concentration of aTC more PTet promoter get free and
more GFP protein produced.
4. Experiment show that the response of the circuits is inversely
proportional to the increase in temperature. Which is consistent with the
mathematical model.

Where, ɑ is maximal production rate and ɣ is degradation rate constant.

On increasing temperature both ɑ and ɣ increases, but increase in ɣ is
significant as compared to ɑ, hence the steady state response of the circuit
decreases with increase in temperature.

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 Chapter-4
4.1 Summary

Robustness to temperature is an important part of designing and

estimation of the nature of bio-molecular circuits. We use negative feedback
circuit to answer this by using mathematical models and experimental
measurements. We observe that both the amplitude and the transient response
changes with the change in temperature. We have examined the mathematical
model of these circuits and see that the parameter of reaction rate facilitate the
temperature dependance,and also we emphasize that some parameter regimes
also increase the temperature robustness but at a performance cost.We
experimentally show that the response of negative feedback facilite temperature
robustness due to these parameter regimes. All these results are very useful in
designing and analysing the temperature robust biomolecular circuits.

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References:
[1] Sen S, Murray RM, “Negative Feedback Facilitates Temperature Robustness
in Biomolecular Circuit Dynamics”, bioRxiv preprint server, 10.1101/007385,
2014
[2] Patel A, Murray RM, Sen S, “Assessment of Robustness to Temperature in a
Negative Feedback Loop and a Feedforward Loop”, bioRxiv preprint server,
10.1101/774042, 2019.
[3] O. Oleksiuk, V. Jakovljevic, N. Vladimirov, R. Carvalho, E. Paster, W. S.
Ryu, Y. Meir, N. S. Wingreen, M. Kollmann, and V. Sourjik. Thermal
robustness of signalling in bacterial chemotaxis. Cell, 145:312–21, 2011.
[4] M. Nakajima, K. Imai, H. Ito, T. Nishiwaki, Y. Murayama, H. Iwasaki, T.
Oyama, and T. Kondo. Reconstitution of circadian oscillation of cyanobacterial
KaiC phosphorylation in vitro. Science, 308(5720):414–5, 2005.
[5] A. B. Reyes, J. S. Pendergast, and S. Yamazaki. Mammalian peripheral
circadian oscillators are temperature compensated. J. Biol. Rhythms, 23:95–98,
2008.
[6] Shaunak Sen and Richard M. Murray. Temperature dependence of
biomolecular circuit designs. In 52nd IEEE Conference on Decision and
Control, pages 1398–1403, December 2013.
[7] Shaunak Sen, Jongmin Kim, and Richard M. Murray. Designing robustness
to temperature in a feedforward loop circuit. In 53rd IEEE Conference on
Decision and Control, pages 4629–4634, December 2014

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