Team:Peking/Model

Peking iGEM 2017

Overview


Background

In our design, after having designed the flip flop, the device can remember the information of its state, the next step is to transform the state into an actual function. To achieve this transformation, we first needed a “reader” to read out the current state. At this point the control unit comes into play. A control unit is a DNA sequence with recombinase sites whose expression is controlled by recom-binase and RDF by reversing or deleting a promoter and/or a terminator. To make the control unit reliable and predictable, we first need to be able to predict the behaviors of its “building blocks” (or “elements” in electrical engineering), from which we weave our engineer’s perspective into the biolog-ical system. However, we need to make some adaptations and adjustments to these “elements” to make them usable.

Flip-flop


Intention of the Model

According to our design of biological flip-flops, a recombinase-based device must be constructed, characterized and standardized. Recombinases can operate on sequences with recombination sites thus altering the direction of transcription. In this section we propose a model that characterize the process of gene recombination with ordinary differential equations (ODEs). Each reaction in the process is described with Hill or mas action kinetics. The aims of this model are listed as follows:
  1. Estimate parameters in the model from experimental data and fine-tune the model.
  2. Simulate the sequence recombination process via recombinases. Show the changing of quantities of original and flipped sequences over time. Prove that the device can work as expected.
  3. Analyze the effects of varying recombinase production and degradation rates on the overall performance of the device.
  4. Assist in designing of the genetic circuit.

Biological Basis

The sequence-flipping device is based on the integrase family of recombinases. On this page we focus only on the Bxb1 serine integrase, but the same dynamics model and analysis applies to two other types TP901 and PhiC31. Mechanism of serine integrases is well recorded in literature.1

SET Process

After transcription and translation of the Bxb1 recombinase gene, the proteins are expressed. Two Bxb1 monomers form a dimer. In dimerized form, the recombinase proteins can bind to attL and attR binding sites around the constitutive promoter J23119 on the reporter sequence. When both sites are occupied, synapse between the two sites can happen. This can enable flipping of the sequence and alter the direction of the promoter. The flipping process changes the direction of the reporter promoter and transforms the attB and attP sites in attL and attR.
In its initial direction, the promoter starts GFP transcription; after flipping, it turns to RFP. By measuring cell fluorescence with flow cytometry, we can quantify the ratio of flipped sequences (attL/R ratio). The data-fitting procedure relies on this key indicator.

RESET Process with Integrase-RDF Fusion Protein

此处应有RDF机理。
The following part describes species and reactions involved in different components of the model.

Model Composition

pBAD – Arabinose-Induced Protein Expression

We have decided to use an arabinose-induced promoter (pBAD) for the expression of recombinases on ColE1 vector. This promoter can be modelled as the following chemical system.



The promoter pBAD binds to AraC and this represses transcription of mRNA. Arabinose will bind to AraC and form the Arab:AraC compound, allowing transcription to occur.

Assumptions

We assume that:
  1. AraC is always in large concentration
  2. AraC binding to arabinose occurs on a faster time scale to transcription
Therefore it is not necessary to consider individual concentrations of arabinose and AraC. We need only include the concentration of (Arab:AraC) complex in the model. The transcription rate constant K on the schematic diagram above is not a simple constant but described as a Michaelis-Mentin kinetics2 equation below.

Although pBAD system is known for rigidity3, we observed leaky expression in the experiments. So we add a basal translation level to describe leakiness. The basal expression level should be several orders of magnitude lower than the maximal level.

Overview


Background

In our design, after having designed the flip flop, the device can remember the information of its state, the next step is to transform the state into an actual function. To achieve this transformation, we first needed a “reader” to read out the current state. At this point the control unit comes into play. A control unit is a DNA sequence with recombinase sites whose expression is controlled by recom-binase and RDF by reversing or deleting a promoter and/or a terminator. To make the control unit reliable and predictable, we first need to be able to predict the behaviors of its “building blocks” (or “elements” in electrical engineering), from which we weave our engineer’s perspective into the biolog-ical system. However, we need to make some adaptations and adjustments to these “elements” to make them usable.

Overview


Background

In our design, after having designed the flip flop, the device can remember the information of its state, the next step is to transform the state into an actual function. To achieve this transformation, we first needed a “reader” to read out the current state. At this point the control unit comes into play. A control unit is a DNA sequence with recombinase sites whose expression is controlled by recom-binase and RDF by reversing or deleting a promoter and/or a terminator. To make the control unit reliable and predictable, we first need to be able to predict the behaviors of its “building blocks” (or “elements” in electrical engineering), from which we weave our engineer’s perspective into the biolog-ical system. However, we need to make some adaptations and adjustments to these “elements” to make them usable.

Overview


Background

In our design, after having designed the flip flop, the device can remember the information of its state, the next step is to transform the state into an actual function. To achieve this transformation, we first needed a “reader” to read out the current state. At this point the control unit comes into play. A control unit is a DNA sequence with recombinase sites whose expression is controlled by recom-binase and RDF by reversing or deleting a promoter and/or a terminator. To make the control unit reliable and predictable, we first need to be able to predict the behaviors of its “building blocks” (or “elements” in electrical engineering), from which we weave our engineer’s perspective into the biolog-ical system. However, we need to make some adaptations and adjustments to these “elements” to make them usable.