Difference between revisions of "Part:BBa K2201373"
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To design a genetic circuit that amplifiers a reporter signal we decided to use an orthogonal RNA-polymerase to the <i>E.coli</i> RNA-polymerase. We decided to use the T3 DNA-polymerase which was 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 one.
 
To design a genetic circuit that amplifiers a reporter signal we decided to use an orthogonal RNA-polymerase to the <i>E.coli</i> RNA-polymerase. We decided to use the T3 DNA-polymerase which was 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 one.
  
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[[File:T--Bielefeld-CeBiTec--SVI-composite-part.png|800px|center|left|<b>Figure 1:</b> Construction of the standard mRFP reporter and the genetic circuit for the amplification of mRFP expression(BBa_K2201373).]]
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[[File:T--Bielefeld-CeBiTec--SVI-composite-part.png|800px|center|left|<b>Figure 1:</b> Construction of the standard mRFP reporter and the genetic circuit for the amplification of mRFP expression(BBa_K2201373).]]<br>
  
 
Figure 1 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 to low to detect and not visible.
 
Figure 1 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 to low to detect and not visible.

Revision as of 16:23, 30 October 2017


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

T3 RNA-Polymerase with an reversed mRFP under T3 RNA-polymerase control for signal strengthening. 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.


Usage and Biology

The T3 RNA-polymerase is highly specific to T3 promoters and orthogonal to the T7 RNA-polymerase. Thus it can be wide-utilized in synthetic biology, even in expression strains which encode for a T7 RNA-polymerase like E. coli BL21. We wanted to use this polymerase to create a reporter for the use in genetic cuircuits to make even weak 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. This causes that the expression level of the target protein is nearly the same as the expression of the fluorescent protein and 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.

In a lot of applications only a weak expression is, or should be, reached. For our project, the expanding of the genetic code, we needed a reliable reporter to detect the expression of the key gene of our selection plasmid. For our selection system it is important that the genes are only expressed on a weak level. When we just placed the CDS of mRFP behind the CDS of this gene, no fluorescence was visible, but through the function of the key gene 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.

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 orthogonal RNA-polymerase to the E.coli RNA-polymerase. We decided to use the T3 DNA-polymerase which was characterised by iGEM Peking 2010. 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 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 one.

Figure 1: Construction of the standard mRFP reporter and the genetic circuit for the amplification of mRFP expression(BBa_K2201373).

Figure 1 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 to low to detect and 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 advantage of this system is easy to explain. If we assume the expression of the gene of interest is realy 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 in 8 seconds. Construct 2 expresses 1 T3 RNA-polymerase every 52 seconds. These polymerases express mRFP with a chain elogation rate with 170 nucleotides per second. So every T3 RNA-polymerase expresses one mRFP every 4.7 seconds. The resulting mRFP expression is shown in Figure 2.