Difference between revisions of "Part:BBa K2201373"

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<partinfo>BBa_K2201373 short</partinfo>
 
<partinfo>BBa_K2201373 short</partinfo>
  
This part contains a T3 RNA-Polymerase with an reversed mRFP under T3 RNA-polymerase control for signal enhancing. It is an improved reporter and a genetic circuit that could report even weak expression levels. This part was created following the model of an amplifier in electrical engineering to intensify an input signal and could be used in a broad range of synthetic biology applications. We used this part for our selection system for the incorporation of non-canonical amino acids
+
This part contains a T3 RNA-Polymerase with an inverted mRFP under T3 RNA-polymerase control for signal enhancing. It is an improved reporter and a genetic circuit that could report even weak expression levels. This part was designedd based on the model of an amplifier in electrical engineering to intensify an existing input signal and could be used in a broad range of synthetic biology applications. We used this part for our selection system for the incorporation of non-canonical amino acids
  
  
 
===Usage and Biology===
 
===Usage and Biology===
The T3 RNA-polymerase is highly specific to T3 promoters and orthogonal to the T7 RNA-polymerase. Thus it can be widely utilized in synthetic biology, even in expression strains which encode for a T7 RNA-polymerase like <i>E. coli</i> BL21.
+
The T3 RNA-polymerase is highly specific to T3 promoters and orthogonal to the T7 RNA-polymerase. Therefore, it can be widely utilized in synthetic biology, even in expression strains which encode for a T7 RNA-polymerase like <i>E. coli</i> BL21. This orthogonality is due to the promoter specifity of the different polymerases.
We wanted to use this polymerase to create a reporter for the use in genetic circuits to make even weak expression visible.
+
We applied this system to enhance a reporter in genetic circuits to make even weak gene expression visible.
 
   
 
   
At the moment fluorescent proteins with an emission wavelength within the visible spectra are used to report expression of the gene of interest. Therefor the CDS of the fluorescent protein is placed behind the CDS of the target protein without a terminator or promoter in between. Due to that, the expression level of the target protein is nearly the same as the expression of the fluorescent protein. The fluorescence of the protein could be used to find out if the gene of interest is translated. This works fine when the expression of the target protein is strong enough to build enough fluorescent protein to generate a strong fluorescent signal.  
+
At the moment, fluorescent proteins with an emission wavelength within the visible spectra are used to report expression of the gene of interest. Therefore, the CDS of the fluorescent protein was placed downstream of the CDS of the target protein without a terminator or promoter in between. The expression level of the target protein is nearly the same as the expression of the fluorescent protein. The fluorescence of the reporter protein indicates if the gene of interest was translated. However, thissystem is limited to strong expression, which generate a sufficienly strong fluorescence signal.  
  
In a lot of applications only a weak expression is, or should be, reached. For our project, we needed a reliable reporter to detect the expression of our gene of interest on our selection plasmid. For our selection system it is important that the genes are only expressed on a weak level. If the CDS of mRFP is placed behind the CDS of this gene, no fluorescence was visible. Through the function of the gene of interest we knew it was expressed. To solve this reporter problem we intended to build a genetic circuit following the model of an amplifier used in electrical engineering.
+
Several applications involve only a weak expression. For our project, we needed a reliable and sensitive reporter to detect the expression of the gene of interest on a selection plasmid. A low expression in this target gene is essential for the selection system. No fluorescence was detectable, when the CDS of mRFP was placed downstream of the gene of interest. Through the function of the gene of interest, we knew it was expressed. To address this reporter challenge we built a genetic circuit following the model of an amplifier used in electrical engineering.
  
Easy amplifiers have already been built by iGEM Cambridge 2009. They build a simple circuit using an activator which leads to the transcription of a reporter gene on a higher level as under the first promoter. To explain their system they used the term Polymerases per second (PoPs). PoPS is the flow of RNA polymerase molecules along DNA (i.e., 'current' for gene expression). The PoPS level is set by the amount of RNA polymerase molecules that pass a specific position on DNA each second. The system is shown in Figure 1 and should enhance the level of input PoPs.
+
Basic amplifiers were previously submitted to the Registry of biological parts e. g. by iGEM Cambridge 2009. They build a simple circuit using an activator, which increased the transcription of a reporter under control of a second promoter. To explain their system they used the term "Polymerases per second" (PoPs). This unit defined as the flow of RNA polymerase molecules over a promoter region per second. The system developed by Cambridge 2009 (Figure 1) could increase the number of PoPs.  
 
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<html>
 
<div class="figure small">
 
<div class="figure small">
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</div>
 
</div>
 
</html>
 
</html>
To improve this system we decided to use an activator which translates the reporter DNA and does not induce the translation. Because a RNA-polymerase, which transcribes the reporter DNA, could transcribe the reporter DNA severel times and enhance the signal even more.<br>
+
To improve this system we decided to use an activator, which translates the reporter DNA and does only induce the translation of the polymerase which then translates the gene of interest. Amplification was achieved by an RNA-polymerase, which transcribes the reporter DNA, multiple tmes and enhances the signal significantly.<br>
  
  
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===Functional Parameters===
 
===Functional Parameters===
  
To design a genetic circuit that amplifiers a reporter signal we decided to use an RNA-polymerase, orthogonal to the native <i>E.coli</i> RNA-polymerase. We used the T3 RNA-polymerase which characterised by iGEM Peking 2010. The T3 RNA-polymerase is highly specific to T3-promoters and orthogonal to the <i>E. coli </i>DNA-polymerases and even to the T7 RNA-polymerase which is very similar. We decided not to use the T7 RNA-polymerase because of it is often used for recombinant expression and we wanted to provide a system which could be used in expression strains like BL21. The construction of our designed composite part and the standard mRFP reporter are shown in Figure 2.
+
To design a genetic circuit that amplifiers a reporter signal we decided to use an RNA-polymerase, orthogonal to the native <i>E. coli</i> RNA polymerase. Therefore, the T3 RNA-polymerase previously characterized by iGEM Peking 2010 was deployed. The T3 RNA polymerase is highly specific to T3-promoters and orthogonal to the <i>E. coli </i>DNA-polymerases and even to the T7 RNA polymerase. We decided not to use the T7 RNA-polymerase, because it is already part of expression strains like BL21 and would therefore prevent the application in such strains. The construction of our designed composite part comprising a standard mRFP reporter is shown in Figure 2.
  
 
[[File:T--Bielefeld-CeBiTec--SVI-composite-part.png|800px|thumb|center|<b>Figure 2:</b> Construction of the standard mRFP reporter and the genetic circuit for the amplification of mRFP expression(BBa_K2201373).]]<br>
 
[[File:T--Bielefeld-CeBiTec--SVI-composite-part.png|800px|thumb|center|<b>Figure 2:</b> Construction of the standard mRFP reporter and the genetic circuit for the amplification of mRFP expression(BBa_K2201373).]]<br>
  
Figure 2 shows two constructions of reporters for gene expression. The first one is the standard reporter for the gene expression of the target gene, using mRFP as reporter. If the gene of interest is expressed, the mRFP is expressed on nearly the same level. Through the detection of the mRFP fluorescence the gene expression of the gene of interest could be detected. This system is suitable for high expression rates, but if the expression of the target  gene is only on a weak expression level, the fluorescence of the expressed mRFP is too low to detect and thus not visible.
+
Figure 2 shows two reporter constructs for gene expression quantification. The first one is the standard reporter for the gene expression of the target gene, using mRFP as reporter gene. If the gene of interest is expressed, the mRFP is expressed at nearly the same level. The mRFP fluorescence reveals the gene expression of the gene of interest. This system is suitable for but also limited to high expression rates. Low, expression of the target  gene is associated with a low expression of mRFP and thus not visible.
  
The second system is our genetic circuit. In this system the CDS of the target gene is in front of a CDS for a T3 DNA-polymerase. Thus the expression of the target gene is nearly on the same level as the expression of the target gene. The expressed T3 RNA-polymerase expresses the mRFP under the control of the T3-promoter.
+
The second system is our genetic circuit consisting of a CDS of the target gene upstream of the CDS for a T3 DNA-polymerase encoding sequence. Therefore, the expression of the reporter gene is nearly on the same level as the expression of the target gene. The expressed T3 RNA polymerase transcripts the mRFP under the control of the T3 promoter.
To demonstrate the advantages of our new construct we modelled the amount of mRFP-transcript for both constructs.
+
To demonstrate the advantages of our improved construct, we modelled the amount of mRFP transcript for both constructs.
If we assume the expression of the gene of interest is low and only one <i>E. coli</i> RNA-polymerase with a chain elongation rate of 50 nucleotides per second translates the both products, construct 1 produces 1 mRFP-transcript in 16 seconds. Construct 2 expresses 1 T3 RNA-polymerase-transcript every 52 seconds. After translation (with a translation rate of 20 amino acids per second ~42 sec), these polymerases transcribe mRFP-transcript with a chain elongation rate of 170 nucleotides per second. So every T3 RNA-polymerase expresses one mRFP-transcript every 4.7 seconds. The resulting amount of mRFP transcripts  is shown in Figure 3. The script for our modelling can be found <html><a href="https://static.igem.org/mediawiki/parts/8/8a/T--Bielefeld-CeBiTec--model-composite-2.txt">here</a></html>.
+
If we assume the expression of the gene of interest is low and only one <i>E. coli</i> RNA polymerase with a chain elongation rate of 50 nucleotides per second translates the both products, construct 1 produces 1 mRFP-transcript in 16 seconds. Construct 2 expresses 1 T3 RNA-polymerase-transcript every 52 seconds. After translation (with an averange  translation rate of 20 amino acids per second ~42 sec), these polymerases transcribe the mRFP transcript with a chain elongation rate of 170 nucleotides per second. Therefore, every T3 RNA polymerase generates one mRFP transcript every 4.7 seconds. The resulting amount of mRFP transcripts  is shown in Figure 3. The script for our modelling can be found <html><a href="https://static.igem.org/mediawiki/parts/8/8a/T--Bielefeld-CeBiTec--model-composite-2.txt">here</a></html>.
  
 
[[File:T--Bielefeld-CeBiTec--model-composite.png|600px|thumb|center|<b>Figure 3:</b> Modelling on the amount of mRFP-transcript transcribed through the two different models.]]<br>
 
[[File:T--Bielefeld-CeBiTec--model-composite.png|600px|thumb|center|<b>Figure 3:</b> Modelling on the amount of mRFP-transcript transcribed through the two different models.]]<br>
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[[File:T--Bielefeld-CeBiTec--composite-1.png|600px|thumb|center|<b>Figure 4:</b> Two Smear of two clones containing only mRFP under an uninduced T7 promoter and containing the mRFP enhancing system under the same promotor, after 12 h of incubation at 37 °C.]]<br>
 
[[File:T--Bielefeld-CeBiTec--composite-1.png|600px|thumb|center|<b>Figure 4:</b> Two Smear of two clones containing only mRFP under an uninduced T7 promoter and containing the mRFP enhancing system under the same promotor, after 12 h of incubation at 37 °C.]]<br>
  
To demonstrate the signal enahncing system we cloned the part E1010 and our signal enhancing system (BBa_K2201373) behind the part BBa_K2201900 containing our positive selection plasmid. A smear of the transformands is shown in Figure 4. The transcription of the promotor is only a basal transcription, thus, it is a weak transcription. Despite this weak transcription, the expressed mRFP of the signal enhancing system is clearly visible, while the fluorescence of only the mRFP is not visible.
+
To demonstrate the signal enahancing system we cloned the part E1010 and our signal enhancing system (BBa_K2201373) downstream of the part BBa_K2201900. This plasmid was characterized in cells containing our positive selection plasmid. A smear of the transformants is shown in Figure 4. This  promoter leads only to a basal transcription. Despite this weak transcription, the expressed mRFP of the signal enhancing system is clearly visible, while the fluorescence of the mRFP in cells without this amplification system is not visible.
  
 
[[File:T--Bielefeld-CeBiTec--composite-2.png|600px|thumb|center|<b>Figure 5:</b> Picture of a negative selection round. The clon still containing the positive selection plasmid, thus the mRFP enhancing system, is red.]]<br>
 
[[File:T--Bielefeld-CeBiTec--composite-2.png|600px|thumb|center|<b>Figure 5:</b> Picture of a negative selection round. The clon still containing the positive selection plasmid, thus the mRFP enhancing system, is red.]]<br>
  
For the selection of tRNA/aminoacylsynthetase for the incorporation of non-canonical amino acids we needed to check if clones still contain the positive selection plasmid in the negative selection round. Therefore, we decided to incorporate the mRFP signal enhancing system behind the CDS of our positive selection plasmid BBa_K2201900. If the cells contain this plasmid in the negative selection, they should be visible as red clones. In contrast, the clones containing the negative selection plasmid should be coulourless. A picture of one round of the negative selection, shown in Figure 5, shows that one clone is red. Thus this clone contains the positive selection plasmid is is a false positive result of the negative selection.
+
For the selection of tRNA/aminoacylsynthetase to incorporate non-canonical amino acids, we needed to check if clones still contain the positive selection plasmid in the negative selection round. Therefore, we decided to incorporate the mRFP signal enhancing system downstream of the CDS of our positive selection plasmid BBa_K2201900. If the cells contain this plasmid in the negative selection, they should be visible as red clones. In contrast, the clones containing the negative selection plasmid should be colorless. A picture of one round of the negative selection (Figure 5) shows that one clone is red, thus demonstrating the function of the system.
  
Apart from our application for this part, there are a lot of applications suitable for this signal enhancing part. Despite all applications where even weak expression should be reported, it is a usefull method which could be transfered to a lot of problems in synthetic biology requiering a stronger output signal.
+
In addition to our application for this part, there are a lot of potential applications for this signal enhancing part. These are by no way limites to reporter signal enhancements.
  
 
<partinfo>BBa_K2201373 parameters</partinfo>
 
<partinfo>BBa_K2201373 parameters</partinfo>

Revision as of 23:17, 31 October 2017


T3 polymerase with inverted mRFP under T3 promoter control for signal enhancing

This part contains a T3 RNA-Polymerase with an inverted mRFP under T3 RNA-polymerase control for signal enhancing. It is an improved reporter and a genetic circuit that could report even weak expression levels. This part was designedd based on the model of an amplifier in electrical engineering to intensify an existing input signal and could be used in a broad range of synthetic biology applications. We used this part for our selection system for the incorporation of non-canonical amino acids


Usage and Biology

The T3 RNA-polymerase is highly specific to T3 promoters and orthogonal to the T7 RNA-polymerase. Therefore, it can be widely utilized in synthetic biology, even in expression strains which encode for a T7 RNA-polymerase like E. coli BL21. This orthogonality is due to the promoter specifity of the different polymerases. We applied this system to enhance a reporter in genetic circuits to make even weak gene expression visible.

At the moment, fluorescent proteins with an emission wavelength within the visible spectra are used to report expression of the gene of interest. Therefore, the CDS of the fluorescent protein was placed downstream of the CDS of the target protein without a terminator or promoter in between. The expression level of the target protein is nearly the same as the expression of the fluorescent protein. The fluorescence of the reporter protein indicates if the gene of interest was translated. However, thissystem is limited to strong expression, which generate a sufficienly strong fluorescence signal.

Several applications involve only a weak expression. For our project, we needed a reliable and sensitive reporter to detect the expression of the gene of interest on a selection plasmid. A low expression in this target gene is essential for the selection system. No fluorescence was detectable, when the CDS of mRFP was placed downstream of the gene of interest. Through the function of the gene of interest, we knew it was expressed. To address this reporter challenge we built a genetic circuit following the model of an amplifier used in electrical engineering.

Basic amplifiers were previously submitted to the Registry of biological parts e. g. by iGEM Cambridge 2009. They build a simple circuit using an activator, which increased the transcription of a reporter under control of a second promoter. To explain their system they used the term "Polymerases per second" (PoPs). This unit defined as the flow of RNA polymerase molecules over a promoter region per second. The system developed by Cambridge 2009 (Figure 1) could increase the number of PoPs.

Figure 1: Signal strenthening system of iGEM Cambridge 2009.

To improve this system we decided to use an activator, which translates the reporter DNA and does only induce the translation of the polymerase which then translates the gene of interest. Amplification was achieved by an RNA-polymerase, which transcribes the reporter DNA, multiple tmes and enhances the signal significantly.


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 2717
    Illegal AgeI site found at 2829
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2585


Functional Parameters

To design a genetic circuit that amplifiers a reporter signal we decided to use an RNA-polymerase, orthogonal to the native E. coli RNA polymerase. Therefore, the T3 RNA-polymerase previously characterized by iGEM Peking 2010 was deployed. The T3 RNA polymerase is highly specific to T3-promoters and orthogonal to the E. coli DNA-polymerases and even to the T7 RNA polymerase. We decided not to use the T7 RNA-polymerase, because it is already part of expression strains like BL21 and would therefore prevent the application in such strains. The construction of our designed composite part comprising a standard mRFP reporter is shown in Figure 2.

Figure 2: Construction of the standard mRFP reporter and the genetic circuit for the amplification of mRFP expression(BBa_K2201373).

Figure 2 shows two reporter constructs for gene expression quantification. The first one is the standard reporter for the gene expression of the target gene, using mRFP as reporter gene. If the gene of interest is expressed, the mRFP is expressed at nearly the same level. The mRFP fluorescence reveals the gene expression of the gene of interest. This system is suitable for but also limited to high expression rates. Low, expression of the target gene is associated with a low expression of mRFP and thus not visible.

The second system is our genetic circuit consisting of a CDS of the target gene upstream of the CDS for a T3 DNA-polymerase encoding sequence. Therefore, the expression of the reporter gene is nearly on the same level as the expression of the target gene. The expressed T3 RNA polymerase transcripts the mRFP under the control of the T3 promoter. To demonstrate the advantages of our improved construct, we modelled the amount of mRFP transcript for both constructs. If we assume the expression of the gene of interest is low and only one E. coli RNA polymerase with a chain elongation rate of 50 nucleotides per second translates the both products, construct 1 produces 1 mRFP-transcript in 16 seconds. Construct 2 expresses 1 T3 RNA-polymerase-transcript every 52 seconds. After translation (with an averange translation rate of 20 amino acids per second ~42 sec), these polymerases transcribe the mRFP transcript with a chain elongation rate of 170 nucleotides per second. Therefore, every T3 RNA polymerase generates one mRFP transcript every 4.7 seconds. The resulting amount of mRFP transcripts is shown in Figure 3. The script for our modelling can be found here.

Figure 3: Modelling on the amount of mRFP-transcript transcribed through the two different models.

The number of mRFP transcripts is a lot higher with construct 2 and this model leaves out the fact that one transcript of the T3 RNA-polymerase transcript could be translated severel times and enhance the signal even more. Despite this model is simplified, it shows the advantages of using the T3 RNA-polymerase for signal enhancing.

Figure 4: Two Smear of two clones containing only mRFP under an uninduced T7 promoter and containing the mRFP enhancing system under the same promotor, after 12 h of incubation at 37 °C.

To demonstrate the signal enahancing system we cloned the part E1010 and our signal enhancing system (BBa_K2201373) downstream of the part BBa_K2201900. This plasmid was characterized in cells containing our positive selection plasmid. A smear of the transformants is shown in Figure 4. This promoter leads only to a basal transcription. Despite this weak transcription, the expressed mRFP of the signal enhancing system is clearly visible, while the fluorescence of the mRFP in cells without this amplification system is not visible.

Figure 5: Picture of a negative selection round. The clon still containing the positive selection plasmid, thus the mRFP enhancing system, is red.

For the selection of tRNA/aminoacylsynthetase to incorporate non-canonical amino acids, we needed to check if clones still contain the positive selection plasmid in the negative selection round. Therefore, we decided to incorporate the mRFP signal enhancing system downstream of the CDS of our positive selection plasmid BBa_K2201900. If the cells contain this plasmid in the negative selection, they should be visible as red clones. In contrast, the clones containing the negative selection plasmid should be colorless. A picture of one round of the negative selection (Figure 5) shows that one clone is red, thus demonstrating the function of the system.

In addition to our application for this part, there are a lot of potential applications for this signal enhancing part. These are by no way limites to reporter signal enhancements.