Difference between revisions of "Part:BBa K4286103"
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<partinfo>BBa_K4286103 short</partinfo>
 
<partinfo>BBa_K4286103 short</partinfo>
  
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2022 SZU-China has designed the second generation of timed suicide switch. The following improvements have been made to the second-generation effector: the MazF expression device has been deleted; high efficiency tetR binding sites have been added.
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2022 SZU-China has designed the second generation of timed suicide switch. Improvements have been made to the tentative timed suicide switch, creating the stable and elegant timed suicide switch2.0. See more about effector2.0 in <html><a href="https://parts.igem.org/Part:BBa_K4286103 ">BBa_K4286103</a></html>.
  
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<center>[图片]</center>
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See more about Oscillator1.0 in <html><a href="https://parts.igem.org/Part:BBa_K4286099 ">BBa_K4286099</a></html> and Effector1.0 in <html><a href="https://parts.igem.org/Part:BBa_K4286100 ">BBa_K4286100</a></html>.
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<center><b>Figure 1. [prb1]-pCAMBIA1302</b></center>
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===Sequencing===
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<!-- -->
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4286103 SequenceAndFeatures</partinfo>
  
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===Usage and Biology===
 
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The core of the effector is the MazEF toxin-antitoxin system from Bacillus subtilis, which can lead to programmed cell death. The antitoxin MazE is controlled by the Promoter tetR, and the toxin MazF is controlled by the constitutive Promoter PJ23110. MazF is an endonuclease that specifically cleaves UACAU sites on mRNA. MazE combines with MazF at a ratio of 1:1 to occupy its active site and make it lose its toxicity.
 
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what's more, the effector2.0 removes the function of periodically expressing MazF and adds the function of binding tetR, acting as a tetR sponge. The effector2.0 is encoded in the high-copy plasmid colE1.
 
  
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===Assembly===
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<!-- Uncomment this to enable Functional Parameter display
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The oscillator2.0 and the effector2.0 form a timed suicide switch2.0.
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===Functional Parameters===
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<partinfo>BBa_K4286103 parameters</partinfo>
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<!-- -->
  
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The engineered bacteria with a timed suicide switch were placed in an IPTG-rich medium or in a dormant state before being applied in fields. The purpose of being placed in IPTG is to continuously activate the PlacI and make the oscillator unbalanced and stagnant， in which circumstance MazF does not express.
+
===Usage and Biology===
  
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After being applied to the field, the oscillator is re-activated with the release of IPTG and the resuscitation of the engineering bacteria. The contents of three repressor proteins changed cyclically: lacI inhibited the expression of tetR, tetR inhibited the expression of λ cI, and λ cI inhibited lacI expression. That is, the three promoters PlacI, PtetR, and PλcI were alternately activated.
+
Effector is the device performing suicide. The core of the effector1.0 is the MazEF toxin-antitoxin system from Bacillus subtilis, which can lead to programmed cell death. The antitoxin MazE is controlled by the Promoter PJ23110, and the toxin MazF is controlled by the constitutive Promoter tetR. MazF is an endonuclease that specifically cleaves UACAU sites on mRNA. MazE combines with MazF at a ratio of 1:1 to occupy its active site and make it lose its toxicity.
  
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As for the effector, MazE was constitutively expressed and maintained at a certain concentration in the cytoplasm, while the expression of MazF was inhibited by tetR and showed a fluctuating increase. In a simplified model, MazE and MazF bind at the ratio of 1:1, resulting in toxin inactivation. When the concentration of toxin MazF is higher than that of antitoxin MazE, the extra toxin MazF plays the role of endonuclease to cut mRNA and kill the engineered microorganisms.
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<center>[[File:K4286101-effector2.png]]</center>
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<center><b>Figure 1. The Effector2.0</b></center>
  
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==Modeling==
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The following improvements have been made to the second-generation effector: the MazF expression device has been deleted; high efficiency tetR binding sites have been added. what's more, the effector2.0 removes the function of periodically expressing MazF and adds the function of binding tetR, acting as a tetR sponge. The effector2.0 is encoded in the high-copy plasmid colE1.
  
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===Assumption===  
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===Assembly===
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In order to establish the oscillator model, the following assumptions are necessary for the model:
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The improved timed suicide switch consists of oscillator2.0 and effector2.0.
  
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Assumption 1: The amount of DNA in the same cell is constant and the physiological behavior is same. The DNA in the same cell can be expressed at the corresponding gene fragment.  
+
The engineered bacteria with a timed suicide switch were placed in an IPTG-rich medium or in a dormant state before being applied in fields. The purpose of being placed in IPTG is to continuously activate the PlacI and make the oscillator unbalanced and stagnant， in which circumstance MazF does not express.
  
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Assumption 2: Transcription and translation processes are carried out under saturation conditions. In the process of transcription and translation, polymerases, ribosomes, amino acids and nucleotides are present in large amounts.  
+
After being applied to the field, the oscillator is re-activated with the release of IPTG and the resuscitation of the engineering bacteria. The contents of three repressor proteins changed cyclically: lacI inhibited the expression of tetR, tetR inhibited the expression of λ cI, and λ cI inhibited lacI expression. That is, the three promoters PlacI, PtetR, and PλcI were alternately activated.
  
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Assumption 3: Degradation of protein and mRNA as well as reactive degradation. That is, proteins and mRNA can be degraded directly without intermediate products.  
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<center>[[File:K4286101-Timed-suicide-switch2.png]]</center>
 +
<center><b>Figure 2. The timed suicide switch2.0</b></center>
  
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Assumption 4: Transcription rate can be modelled by Hill equation. That is because the transcription rate of each gene is determined by the concentration of its protein product, and the repressor protein binds to the regulatory region of the gene faster than transcription and translation.
 
  
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Assumption 5: The Hill coefficient approximates the number of cooperative ligand binding sites on the receptor, and ligand molecules bind to a receptor simultaneously.  
+
As for the effector, MazE was constitutively expressed and maintained at a certain concentration in the cytoplasm, while the expression of MazF was inhibited by tetR and showed a fluctuating increase. In a simplified model, MazE and MazF bind at the ratio of 1:1, resulting in toxin inactivation. When the concentration of toxin MazF is higher than that of antitoxin MazE, the extra toxin MazF plays the role of endonuclease to cut mRNA and kill the engineered microorganisms.
  
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Assumption 6: The translation rate of each gene is equal.
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===Reasons for improvement===
 +
<b>1. The number of effector plasmids fluctuates at a high level</b>
  
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===Modeling===
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First, in the timed suicide switch1.0, the oscillator is placed on a rigor type plasmid and the effector is placed on a relaxation type plasmid. The two devices are in a separated state and communicate with each other through tetR (tet repressor). Due to the poor replication control of effector plasmids used, the number of effector plasmids fluctuated at a high level, and the number of effector plasmids in each cell of the engineered bacteria population was not strictly the same. This difference sets the activation and inhibition of Promoter tetR on the oscillator and effector at variance, making it difficult to achieve synchronous oscillation of toxin mazF's concentration in the population.
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The expression processes of the 3 genes lacI,tetR and cI are described as follows:
+
  
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<center>[图片]</center>
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Therefore, we transferred the MazF expression device from the effector plasmid to the low-copy oscillator plasmid, which could greatly reduce the amplitude standard deviation of MazF oscillation and help to achieve the synchronization of engineering bacteria.
  
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For the transcription process, the ODE for transcription rate is described as follows:
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<b>2.Overloading of Proteolytic System in engineering bacteria</b>
  
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<center>[图片]</center>
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Secondly, for the purpose of realizing the rapid oscillation, the C-terminal of the three repressor proteins of the oscillator in the timed suicide switch1.0 is added with a rapid degradation tag. However, because these repressor proteins with the tag share the same Proteolytic System with other proteins, this leads to an overload. Theory shows that the supersaturation of the Proteolytic System will lead to large random fluctuations in the oscillation of individual cell, and the fluctuation caused by the delayed degradation of repressor will be fed back to the repressor protein through the Proteolytic System, thus interfering with the oscillation.
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+
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Hill equation：
+
  
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<center>[图片]</center>
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Therefore, we deleted the LVA degradation tag at the C-terminus of repressor proteins, eliminating the degradation competition between repressor proteins, which will reduce the noise of single cell oscillation and prolong the oscillation period. This will also extend the suicide time of engineered bacteria, so that the engineered bacteria can survive longer, which is good for our project.
  
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To carry out dimension reduction and simplify the analysis of this system, we normalize the ODEs
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<b>3. Low disinhibition prefabricated of Promoter tetR</b>
  
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===Results===
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Third, previous studies have shown that the noisiest phase of oscillations is when the level of TetR is the lowest. At this time, the disinhibition of Promoter tetR occurs at a very low threshold of tetR. Theory suggests that if this threshold is raised, the regularity of oscillations may be greatly improved.
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We set the oscillator model parameters as:
+
  
−
<center>[图片]</center>
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We added five highly efficient tetR binding sites to the effector plasmid. The modified effector plasmid can be used as a sponge of tetR to adsorb tetR. The tetR binding site was derived from Promoter tetR and artificially engineered to increase binding efficiency. In theory, the introduction of tetR molecular sponge will reduce the effective concentration of tetR in the cytoplasm, thus increasing the apparent threshold of Promoter tetR disinhibition and greatly improving the regularity of the oscillator.
  
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First, we set the initial number of mRNA molecules and the number of protein molecules to be both 0. The oscillator model was simulated for 1000 minutes. Note that only the curve representing the number of cI protein molecules is visible, as all plotted concentrations are identical and overlap. From the simulation results, it can be seen that the peak value is 288 molecules at 8 minutes, and the steady state is reached at 28 minutes, where the number of protein molecules is maintained at 210 molecules.
+
===Prediction===
 +
In theory, the oscillating rise of MazF concentration and the stable concentration of MazE can be predicted by the mathematical model. That is, we can predict the exact  time point of suicide as long as we have accurate parameters. We performed a modeling analysis of the timed suicide switch1.0, mathematically demonstrating that the engineered bacteria will begin to suicide after approximately 770.1 minutes, when the concentration of toxin mazF is higher than that of antitoxin MazE reversely.
  
−
<center>[图片]</center>
+
<center>[[File:K4286101-Prediction.png]]</center>
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<center><b>Figure 1. [prb1]-pCAMBIA1302</b></center>
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<center><b>Figure 3. The prediction of timed suicide switch</b></center>
  
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We set the initial mRNA molecules and protein molecules to $$m_{lacI}(t = 0)=m_{tetR}(t = 0) = m_ {cI} (t = 0) = 0$$, $$p_ {tetR} = 10, p_ {lacI} = p_ {cI} = 0 $$, The oscillator model was then simulated for 1000 minutes, and the simulation results are shown in Figure 12. It is clear that after a period of time in the oscillator system, the system reaches a steady state with the peak value is 3000 molecules of the 3 proteins and the peak-to-peak period is 175 min.
+
Furthermore, we may also be able to achieve different suicide time by changing the parts of the gene device. For example, by replacing the constitutive promoter with different transcriptional activity, we can change the stable concentration of antitoxin MazE, so as to change the time of concentration reversal of MazF and MazE. Thereby changing the suicide time of the engineering microorganisms.
  
−
<center>[图片]</center>
+
See more about the model of timed suicide switch1.0 in <html><a href="https://parts.igem.org/Part:BBa_K4286099 ">BBa_K4286099</a></html>.
−
<center><b>Figure 1. [prb1]-pCAMBIA1302</b></center>
+
  
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It is clear that after a period of time in the oscillator system, the system reaches a steady state with the peak value is 3000 molecules of the 3 proteins and the peak-to-peak period is 175 min.
+
===References===
 +
[1]Elowitz MB, Leibler S. A synthetic oscillatory network of transcriptional regulators. Nature. 2000 Jan 20;403(6767):335-8. doi: 10.1038/35002125. PMID: 10659856.
  
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In order to change the peak amplitude of the oscillator model and the peak when the steady state is reached, we also explored the effect of different initial conditions on the oscillator model. In addition, we used 3D representation that plotted the simulation of the oscillator system for 1000 minutes (Figure 13), to fully understand the behavior of the oscillator system. Figure 13(a). shows that the system will rapidly reach the steady state under different initial protein numbers for 1000 minutes, unless all concentrations are the same, the system will begin to approach the limit cycle, and the steady state simulation is shown in red. Figure 13(b). shows the changes in TetR and LacI protein concentrations during the simulation. Figure 13(c). shows the curve of the region near the steady state near the limit cycle of Figure 13(b). Figure 13(d). shows that when the oscillator system is greater than 800 minutes, except for the steady state (indicated in red), it is related to the limit cycle. Figure 13(e). shows that for more than 200 minutes, the closer the initial conditions are to the same, the longer it takes for the system to reach the limit cycle.
+
[2]Purcell O, di Bernardo M, Grierson CS, Savery NJ. A multi-functional synthetic gene network: a frequency multiplier, oscillator and switch. PLoS One. 2011 Feb 17;6(2):e16140. doi: 10.1371/journal.pone.0016140. PMID: 21359152; PMCID: PMC3040778.
  
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<center>[图片]</center>
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[3]Potvin-Trottier L, Lord ND, Vinnicombe G, Paulsson J. Synchronous long-term oscillations in a synthetic gene circuit. Nature. 2016 Oct 27;538(7626):514-517. doi: 10.1038/nature19841. Epub 2016 Oct 12. PMID: 27732583; PMCID: PMC5637407.
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<center><b>Figure 1. [prb1]-pCAMBIA1302</b></center>
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===Sequencing===
+
[4]Hoseini S, Kalani BS, Ghafourian S, Maleki A, Asadollahi P, Badakhsh B, Pakzad I. In Vitro and In Silico Investigation of some Type II TA Genes in H. Pylori. Clin Lab. 2022 Aug 1;68(8). doi: 10.7754/Clin.Lab.2021.211002. PMID: 35975492.
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<!-- -->
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<span class='h3bb'>Sequence and Features</span>
+
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<partinfo>BBa_K4286103 SequenceAndFeatures</partinfo>
+
  
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+
[5]Nigam A, Ziv T, Oron-Gottesman A, Engelberg-Kulka H. Stress-Induced MazF-Mediated Proteins in Escherichia coli. mBio. 2019 Mar 26;10(2):e00340-19. doi: 10.1128/mBio.00340-19. PMID: 30914510; PMCID: PMC6437054.
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
+
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<partinfo>BBa_K4286103 parameters</partinfo>
+
−
<!-- -->
+

Latest revision as of 14:49, 12 October 2022


Effector device for improved version of timed suicide switch

2022 SZU-China has designed the second generation of timed suicide switch. Improvements have been made to the tentative timed suicide switch, creating the stable and elegant timed suicide switch2.0. See more about effector2.0 in BBa_K4286103.

See more about Oscillator1.0 in BBa_K4286099 and Effector1.0 in BBa_K4286100.

Sequencing

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 108
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Effector is the device performing suicide. The core of the effector1.0 is the MazEF toxin-antitoxin system from Bacillus subtilis, which can lead to programmed cell death. The antitoxin MazE is controlled by the Promoter PJ23110, and the toxin MazF is controlled by the constitutive Promoter tetR. MazF is an endonuclease that specifically cleaves UACAU sites on mRNA. MazE combines with MazF at a ratio of 1:1 to occupy its active site and make it lose its toxicity.

K4286101-effector2.png
Figure 1. The Effector2.0

The following improvements have been made to the second-generation effector: the MazF expression device has been deleted; high efficiency tetR binding sites have been added. what's more, the effector2.0 removes the function of periodically expressing MazF and adds the function of binding tetR, acting as a tetR sponge. The effector2.0 is encoded in the high-copy plasmid colE1.

Assembly

The improved timed suicide switch consists of oscillator2.0 and effector2.0.

The engineered bacteria with a timed suicide switch were placed in an IPTG-rich medium or in a dormant state before being applied in fields. The purpose of being placed in IPTG is to continuously activate the PlacI and make the oscillator unbalanced and stagnant， in which circumstance MazF does not express.

After being applied to the field, the oscillator is re-activated with the release of IPTG and the resuscitation of the engineering bacteria. The contents of three repressor proteins changed cyclically: lacI inhibited the expression of tetR, tetR inhibited the expression of λ cI, and λ cI inhibited lacI expression. That is, the three promoters PlacI, PtetR, and PλcI were alternately activated.

K4286101-Timed-suicide-switch2.png
Figure 2. The timed suicide switch2.0


As for the effector, MazE was constitutively expressed and maintained at a certain concentration in the cytoplasm, while the expression of MazF was inhibited by tetR and showed a fluctuating increase. In a simplified model, MazE and MazF bind at the ratio of 1:1, resulting in toxin inactivation. When the concentration of toxin MazF is higher than that of antitoxin MazE, the extra toxin MazF plays the role of endonuclease to cut mRNA and kill the engineered microorganisms.

Reasons for improvement

1. The number of effector plasmids fluctuates at a high level

First, in the timed suicide switch1.0, the oscillator is placed on a rigor type plasmid and the effector is placed on a relaxation type plasmid. The two devices are in a separated state and communicate with each other through tetR (tet repressor). Due to the poor replication control of effector plasmids used, the number of effector plasmids fluctuated at a high level, and the number of effector plasmids in each cell of the engineered bacteria population was not strictly the same. This difference sets the activation and inhibition of Promoter tetR on the oscillator and effector at variance, making it difficult to achieve synchronous oscillation of toxin mazF's concentration in the population.

Therefore, we transferred the MazF expression device from the effector plasmid to the low-copy oscillator plasmid, which could greatly reduce the amplitude standard deviation of MazF oscillation and help to achieve the synchronization of engineering bacteria.

2.Overloading of Proteolytic System in engineering bacteria

Secondly, for the purpose of realizing the rapid oscillation, the C-terminal of the three repressor proteins of the oscillator in the timed suicide switch1.0 is added with a rapid degradation tag. However, because these repressor proteins with the tag share the same Proteolytic System with other proteins, this leads to an overload. Theory shows that the supersaturation of the Proteolytic System will lead to large random fluctuations in the oscillation of individual cell, and the fluctuation caused by the delayed degradation of repressor will be fed back to the repressor protein through the Proteolytic System, thus interfering with the oscillation.

Therefore, we deleted the LVA degradation tag at the C-terminus of repressor proteins, eliminating the degradation competition between repressor proteins, which will reduce the noise of single cell oscillation and prolong the oscillation period. This will also extend the suicide time of engineered bacteria, so that the engineered bacteria can survive longer, which is good for our project.

3. Low disinhibition prefabricated of Promoter tetR

Third, previous studies have shown that the noisiest phase of oscillations is when the level of TetR is the lowest. At this time, the disinhibition of Promoter tetR occurs at a very low threshold of tetR. Theory suggests that if this threshold is raised, the regularity of oscillations may be greatly improved.

We added five highly efficient tetR binding sites to the effector plasmid. The modified effector plasmid can be used as a sponge of tetR to adsorb tetR. The tetR binding site was derived from Promoter tetR and artificially engineered to increase binding efficiency. In theory, the introduction of tetR molecular sponge will reduce the effective concentration of tetR in the cytoplasm, thus increasing the apparent threshold of Promoter tetR disinhibition and greatly improving the regularity of the oscillator.

Prediction

In theory, the oscillating rise of MazF concentration and the stable concentration of MazE can be predicted by the mathematical model. That is, we can predict the exact time point of suicide as long as we have accurate parameters. We performed a modeling analysis of the timed suicide switch1.0, mathematically demonstrating that the engineered bacteria will begin to suicide after approximately 770.1 minutes, when the concentration of toxin mazF is higher than that of antitoxin MazE reversely.

K4286101-Prediction.png
Figure 3. The prediction of timed suicide switch

Furthermore, we may also be able to achieve different suicide time by changing the parts of the gene device. For example, by replacing the constitutive promoter with different transcriptional activity, we can change the stable concentration of antitoxin MazE, so as to change the time of concentration reversal of MazF and MazE. Thereby changing the suicide time of the engineering microorganisms.

See more about the model of timed suicide switch1.0 in BBa_K4286099.

References

[1]Elowitz MB, Leibler S. A synthetic oscillatory network of transcriptional regulators. Nature. 2000 Jan 20;403(6767):335-8. doi: 10.1038/35002125. PMID: 10659856.

[2]Purcell O, di Bernardo M, Grierson CS, Savery NJ. A multi-functional synthetic gene network: a frequency multiplier, oscillator and switch. PLoS One. 2011 Feb 17;6(2):e16140. doi: 10.1371/journal.pone.0016140. PMID: 21359152; PMCID: PMC3040778.

[3]Potvin-Trottier L, Lord ND, Vinnicombe G, Paulsson J. Synchronous long-term oscillations in a synthetic gene circuit. Nature. 2016 Oct 27;538(7626):514-517. doi: 10.1038/nature19841. Epub 2016 Oct 12. PMID: 27732583; PMCID: PMC5637407.

[4]Hoseini S, Kalani BS, Ghafourian S, Maleki A, Asadollahi P, Badakhsh B, Pakzad I. In Vitro and In Silico Investigation of some Type II TA Genes in H. Pylori. Clin Lab. 2022 Aug 1;68(8). doi: 10.7754/Clin.Lab.2021.211002. PMID: 35975492.

[5]Nigam A, Ziv T, Oron-Gottesman A, Engelberg-Kulka H. Stress-Induced MazF-Mediated Proteins in Escherichia coli. mBio. 2019 Mar 26;10(2):e00340-19. doi: 10.1128/mBio.00340-19. PMID: 30914510; PMCID: PMC6437054.