Team:Technion/Project/Overview

From 2012.igem.org

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(The fluorescent proteins)
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{{:Team:Technion/Project}}
{{:Team:Technion/Project}}
==The general idea==
==The general idea==
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Figure 1 presents the basic strategy for our three inputs AND gate. Each one of the inducers can be an environmental signal, to which the phage will respond. In order to check that the inducers work, we chose to use fluorescent proteins (FPs). The inducers also trigger the production of RNA polymerases (RNAPs), in a logical operation similar to a YES gate. Finally, in the presence of all the inputs, the phage will be capable to resume its lytic cycle, resulting in our AND gate's output.
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Figure 1 presents the basic strategy for our three inputs AND gate. Each one of the inducers can be an environmental signal, to which the phage will respond. In order to check that the inducers work, we chose to use fluorescent proteins (FPs). The inducers also trigger the production of RNA polymerases (RNAPs), in a logical operation similar to a [https://2012.igem.org/Team:Technion/Project/YES_gates#What.27s_a_YES_gate.3F YES gate]. Finally, in the presence of all the inputs, the phage will be capable to resume its lytic cycle, resulting in our AND gate's output.
[[File:Technion overview.jpg]]
[[File:Technion overview.jpg]]
==The inducers, promoters and bacterial strain==
==The inducers, promoters and bacterial strain==
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When considering the choice of our system's components, the choice of the inducers, promoters and bacterial strain is intertwined. We needed to minimize the number of plasmids used, therefore, the bacterial strain should provide us with as many regulatory overexpressed components as possible. Roee had the strain DH5αZ1, which expresses high levels of <i>Lac</i> and <i>Tet</i> repressor (<i>Lac</i>R, <i>Tet</i>R) as well as of AraC {1}. This allowed us to use the P<sub>TetO</sub> and the P<sub>Lac/Ara</sub> promoters without the introduction of additional regulatory elements on plasmids. Consequentially, the inducers used were anhydrotetracycline (aTc) for P<sub>TetO</sub> and IPTG with arabinose for P<sub>Lac/Ara</sub>.<br>
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The inducers serve as the inputs for the three [https://2012.igem.org/Team:Technion/Project/YES_gates#What.27s_a_YES_gate.3F YES gates]. When considering the choice of our system's components, the choice of the inducers, promoters and bacterial strain is intertwined. We needed to minimize the number of plasmids used, therefore, the bacterial strain should provide us with as many regulatory overexpressed components as possible. Roee had the strain DH5αZ1, which expresses high levels of <i>Lac</i> and <i>Tet</i> repressor (<i>Lac</i>R, <i>Tet</i>R) as well as of AraC {1}. This allowed us to use the P<sub>TetO</sub> and the P<sub>Lac/Ara</sub> promoters without the introduction of additional regulatory elements on plasmids. Consequentially, the inducers used were anhydrotetracycline (aTc) for P<sub>TetO</sub> and IPTG with arabinose for P<sub>Lac/Ara</sub>.<br>
The choice of the third promoter could no longer rely on host strain expression of required regulatory elements. Therefore, we chose the quorum sensing system based on the P<sub>Lux</sub> promoter, which is induced by [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] (also known as N-(β-ketocaproyl)-L-Homoserine lactone) in the presence of the LuxR protein. This made it obligatory for us to express the LuxR gene from a plasmid. Moreover, since the P<sub>Lux</sub> promoter is leaky, we chose to introduce another regulatory element to reduce the basal level. This element is the [https://2012.igem.org/Team:Technion/Project/RS theophylline induced riboswitch (RS)], which controls the level of translation of a given protein. This construct is later notated as P<sub>Lux</sub>+RS. More about these elements can be found in the [https://2012.igem.org/Team:Technion/Project/YES_gates#YES_gate_.233 YES gate #3] section.
The choice of the third promoter could no longer rely on host strain expression of required regulatory elements. Therefore, we chose the quorum sensing system based on the P<sub>Lux</sub> promoter, which is induced by [http://partsregistry.org/3OC6HSL 3OC<sub>6</sub>HSL] (also known as N-(β-ketocaproyl)-L-Homoserine lactone) in the presence of the LuxR protein. This made it obligatory for us to express the LuxR gene from a plasmid. Moreover, since the P<sub>Lux</sub> promoter is leaky, we chose to introduce another regulatory element to reduce the basal level. This element is the [https://2012.igem.org/Team:Technion/Project/RS theophylline induced riboswitch (RS)], which controls the level of translation of a given protein. This construct is later notated as P<sub>Lux</sub>+RS. More about these elements can be found in the [https://2012.igem.org/Team:Technion/Project/YES_gates#YES_gate_.233 YES gate #3] section.
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==The RNA polymerases and promoters==
==The RNA polymerases and promoters==
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The output from each of our [https://2012.igem.org/Team:Technion/Project/YES_gates#What.27s_a_YES_gate.3F YES gates] is the production of a distinct RNA polymerase. When choosing the polymerases for our system, a few key features were kept in mind:<br>
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<ol>
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<li> <b>Orthogonality</b> - each of the RNAPs used should be orthogonal to the other components of the circuit. Crosstalk between the different RNAPs should be minimal. Thus, reducing the chance of a false positive result.</li>
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<li> <b>Host compatibility</b> - the chosen RNAPs should be compatible with our chosen host strain on several accounts. Firstly, there should be no crosstalk between the host RNAPs and the chosen promoters. Moreover, the chosen RNAPs should not burden the cell or be toxic to it.</li>
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<li> <b>Tuneable strength</b> - the chosen RNAPs trigger expression from dedicated promoters. It should be possible to tune the level of expression by choosing a specific promoter from a promoter library corresponding to each RNAP</li>
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</ol>
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In order to answer the above demands, bacteriophage RNAPs and promoter libraries were chosen. More information about the different RNAPs can be found in the [https://2012.igem.org/Team:Technion/Project/RNAPs RNAPs section].
==The phage==
==The phage==

Revision as of 11:37, 23 September 2012



Contents

The general idea

Figure 1 presents the basic strategy for our three inputs AND gate. Each one of the inducers can be an environmental signal, to which the phage will respond. In order to check that the inducers work, we chose to use fluorescent proteins (FPs). The inducers also trigger the production of RNA polymerases (RNAPs), in a logical operation similar to a YES gate. Finally, in the presence of all the inputs, the phage will be capable to resume its lytic cycle, resulting in our AND gate's output. Technion overview.jpg

The inducers, promoters and bacterial strain

The inducers serve as the inputs for the three YES gates. When considering the choice of our system's components, the choice of the inducers, promoters and bacterial strain is intertwined. We needed to minimize the number of plasmids used, therefore, the bacterial strain should provide us with as many regulatory overexpressed components as possible. Roee had the strain DH5αZ1, which expresses high levels of Lac and Tet repressor (LacR, TetR) as well as of AraC {1}. This allowed us to use the PTetO and the PLac/Ara promoters without the introduction of additional regulatory elements on plasmids. Consequentially, the inducers used were anhydrotetracycline (aTc) for PTetO and IPTG with arabinose for PLac/Ara.
The choice of the third promoter could no longer rely on host strain expression of required regulatory elements. Therefore, we chose the quorum sensing system based on the PLux promoter, which is induced by [http://partsregistry.org/3OC6HSL 3OC6HSL] (also known as N-(β-ketocaproyl)-L-Homoserine lactone) in the presence of the LuxR protein. This made it obligatory for us to express the LuxR gene from a plasmid. Moreover, since the PLux promoter is leaky, we chose to introduce another regulatory element to reduce the basal level. This element is the theophylline induced riboswitch (RS), which controls the level of translation of a given protein. This construct is later notated as PLux+RS. More about these elements can be found in the YES gate #3 section.

The fluorescent proteins

The purpose of the fluorescent proteins in our system was to verify induction of the various promoters by their inducers. The choice of compatible fluorescent proteins for simultaneous visualization can be tricky. Based on the work done by Tsien, et al {2} we initially chose to use mCitrine (a yellow FP), Cerulean (a cyan FP) and mCherry (a red FP) assigned to PTetO, PLac/Ara and PLux+RS respectively. Following different consideration which are mentioned in the YES gates section for each system. Unfortunately, the mCitrine, which we acquired from Roee, failed to show fluorescence. Therefore, we tried several alternatives which are described in the YES gate #2 section.

The RNA polymerases and promoters

The output from each of our YES gates is the production of a distinct RNA polymerase. When choosing the polymerases for our system, a few key features were kept in mind:

  1. Orthogonality - each of the RNAPs used should be orthogonal to the other components of the circuit. Crosstalk between the different RNAPs should be minimal. Thus, reducing the chance of a false positive result.
  2. Host compatibility - the chosen RNAPs should be compatible with our chosen host strain on several accounts. Firstly, there should be no crosstalk between the host RNAPs and the chosen promoters. Moreover, the chosen RNAPs should not burden the cell or be toxic to it.
  3. Tuneable strength - the chosen RNAPs trigger expression from dedicated promoters. It should be possible to tune the level of expression by choosing a specific promoter from a promoter library corresponding to each RNAP

In order to answer the above demands, bacteriophage RNAPs and promoter libraries were chosen. More information about the different RNAPs can be found in the RNAPs section.

The phage

References

1. Lutz R., Bujard H. 1997. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Research 25(6): 1203-1210.
2. Shaner N. C., Steinbach P. A., Tsien R. Y. 2005. A guide to choosing fluorescent proteins. Nature Methods 2(12): 905-909.