Part:BBa_K598002

Bistable Switch Mutant 68

Description

This part is one of the mutation libraries of bistable switch modifying the ribosome binding site (RBS) of cI434 gene.

The bistable switch, which was inherited from the iGEM 2007 Peking Team, mainly consists of a positive feedback loop and a double-negative feedback loop. The expression of two mutually repressing repressors genes cI434 and cI are controlled by the promoters PR and PRM respectively. Promoter RM can be activated by CI and repressed by CI434. GFP and mRFP are placed downstream cI434 and cI as reporters of two states, respectively. In the state when CI is dominant, it can activate its own gene’s transcription and repress that of cI434, thus developing and stabilizing a stable high CI/low CI434 state, in which a red fluorescent protein (mRFP) gene co-transcripted with CI is expressed. Alternatively, when CI434 is dominant, a stable high CI434/low CI state will be established and GFP co-transcripted with CI434 is expressed to represent the this state. Each cell that bears the bistable switch is expected to express GFP or mRFP exclusively. The two states are believed to be stabilized over a long period while under certain circumstances one state may be turned over to the other.

When the bistable switch on pSB1C3 plasmid is transformed into DH5α strain, green colonies and red colonies could be observed. Interestingly, several mixed colonies could also be observed, which implied the random steady-state characteristic of the bistable switch (Figure 1). A ratiometric of the green colonies to the red colonies (G/R ratio) was calculated on the LB agar plate. We proposed that the G/R ratio is relevant to the translation strength of CI & CI434 genes, which means modulating the translation strength of one or more could result in different ratios of G/R under current architecture of bistable switch.

Figure 1 Images of colonies and individual cells bearing the genetic bistable switch. (A) A red colony and a mixed colony captured by fluorescence stereomicroscope. (B) Individual cell images in the mixed colony captured by laser confocal microscope. Each rod represents a single cell, expressing GFP or mRFP exclusively.

Computational Model

We first demonstrated through mathematical modeling that the genetic device we’ve constructed is indeed able to display properties characteristic of a bistable switch within a particular parameter range. Since we are focusing on the translation level, the only variable will be the translation strength of the gene-of-interest (In our case, CI434, with reasons stated below). The original construct did not display a bistable property because the promoter and the strength of the cI434 gene is too strong compared to that of cI, rendering the system mono-stable in the high CI434/low CI state. To endow the system with bistability, the strength of cI434 production can be down-regulated by reducing translation strength. To quantitatively describe the system, we employed a set of ordinary differential equations (ODEs) to represent the transcription and translation control of the system (See Appendix [1]) . Taking into consideration the stochastic nature of the system [2], we used a stochastic algorithm to produce the probability distribution of the system in every possible state (Figure 2).If the system is a monostable one (e.g., high CI434/low CI), its states will be tightly distributed around a single peak value. As the translation strength of cI434 gradually decreases, the peak falls and another peak indicating the high CI/low CI434 state starts to grow, until the first peak disappears and high CI/low CI434 becomes the predominant state, bringing the system back to a monostable one.

Figure 2 Changes in distribution of the number of CI434 molecules in response to changes in translation strength (βSdcro, a parameter in the ODE model that describes translation rate of cI434 gene. For more details, see Appendix). The surface was generated using a stochastic algorithm that simulates the behavior of 1000 cells. Z-axis (height of the surface) corresponds to the proportion of cells that express a certain number of CI434 molecules. It can be seen that when translation rate is low, the system’s state is tightly distributed around a low-CI434 state, i.e., the system is monostabe. As translation strength grows higher, another distribution peak starts to appear, indicating a high-CI434 state. The system thus enters the bistable region. When translation rate is too high (βSdcro above 0.6), the system returns to a monostable state again, with only the high-CI434 distribution peak.

Library Construction

The bistable switch library modifying the expression of cI434 gene is constructed via site-directed mutagenesis method.(Figure 3)

Figure 3 Construction of bistable switch library via site-directed mutagenesis. Forward and reverse primers binding sites are schematized.

Primers are designed pairing the template (BBa_K228003) but replacing the RBS of cI434 gene with NNNNNNN. After PCR amplification, the PCR product was treated with DpnI and column-purified. The linear DNA was phosphoryated by T4 polynucleotide kinase and column-purified. The DNA was then self-ligated and transformed into DH5α cells. Thus, the library is established.

Experimental Data

This part was experimentally characterized together with other sequences in the mutation library used in the hardcoding process in contrast with our softcoding methodology. The resulting proportion of "green"(high CI434/low CI) states was determined and plotted in a diagram displaying both results from the mutation library and that from the stochastic simulation of 1000 cells.(Figure 4)

Figure 4 Proportion of cells in the high CI434/low CI(green) state derived from data in Figure 3 and experimental results. The black data points were derived by counting the cells in the arbitrarily defined “green” state(cells with more than 300 CI434 molecules) under each translation rate. It can be clearly seen that as translation strength(Arbitrary △G values determined from ODE parameters and normalized to computationally determined values from the mutation library) increases, the system travels from a low CI434, monostable state to a transitional, bistable state and then to a high CI434, monostable state. The simulation data is normalized and fitted to experimental results of hardcoding(site-directed mutagenesis), shown in red data points(with error bar). The two sets of data showed fair fitness with each other, validating our previous assertion that translation strength regulates the device’s behavior. Data point for our favorite part, Bistable Switch Mutant 68, is represented by the blue dot in the red curve. It can be seen that it showed good parallelism in experiment repeats

A fluorescence stereomicroscopic image of the colonies formed from E.coli transformed with this part was acquired to show the bistability of the device. Green and red colonies correspond to those in the high CI434/low CI and high CI/low CI434 states, respectively. This is a result of stochasticity in the initial states of individual cells. Since its translation rate for cI434 gene has been down-regulated through modification of the RBS sequence, it showed satisfactory bistable switch performance.(Figure 5)

Figure 5 Experimental results of part Bistable Switch Mutant 68. Image was acquired by applying excitation light with wavelengths of 470nm(for eGFP) and 580nm(for mRFP) to the agar plate respectively and merging two emission images together. Round or oval bright regions indicate colonies.

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Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 10
  Illegal AgeI site found at 122
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2699