Part:BBa_K2201373
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 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.
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.
Easy amplifiers have already been build by iGEM Cambridge 2009. They builded a simple circuit using an activator which leads to the transcription of a gene in a higher expression level as under the first promoter, both descriped as 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.
Figure 1: Signal strenthening system of iGEM Cambridge 2009.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2717
Illegal AgeI site found at 2829 - 1000INCOMPATIBLE 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 RNA-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 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 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. To demonstrate the advantages of our new constuct we modelled the amount of mRFP-transcript for both constructs. 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-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 elogation 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 2. The script for our modelling could be found here.
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 severell times and enhance the signal even more. Of course this model is simplified, but it shows the advantages of using the T3 RNA-polymerase for signal enhancing.
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