Short SummaryFor the application of our best composite part, we decided to nominate our reporter signal enhancing system BBa_K2201373. This part contains a T3 RNA Polymerase with an inverted mRFP under T3 RNA polymerase control for the enhancing of reporter signals. It is an improved reporter and a genetic circuit that could report even weak expression levels. This part was designed 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 and demonstrate the advantages of our system in comparison with standard reporter in an integrated modelling and wet lab characterizations.
Usage and Biology
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 is 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, this system is limited to strong expression, which generate a sufficienly strong fluorescence signal.
Therefore it is impractical for several applications which involve only 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 of the 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.
Figure 2: Construction of the standard mRFP reporter and the genetic circuit for the amplification of mRFP expression. In construct 1 the CDS fot the mRFP transcript is downstream the CDS of the gene of interest. In construct 2 the CDS of the mRFP is downstream a T3 promoter and the CDS coding for the T3 RNA polymerase is downstream the CDS of the gene of interest.
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 transcribes 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 average 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:Modeling on the amount of mRFP transcript transcribed through the two different models. In model 1 mRFP CDS lies is of the CDS of the gene of interest. In model 2 the CDS of the mRFP is downstream a T3 promoter and the CDS coding for the T3 RNA polymerase is downstream the CDS of the gene of interest.
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.
Figure 5: Picture of a negative selection round. The clone still containing the positive selection plasmid, thus the mRFP enhancing system, is red.
Figure 6: Potential applications for the reporter signal enhancing system.
|BBa_K2201373||Composite||T3 Polymerase with inverted mRFP under T3 promoter control for signal enhancing||Svenja Vinke||3411|
|BBa_K2201201||Composite||Pyrrosyl tRNA/aminoacyl-synthetase for the incorporation of propargyllysine||Svenja Vinke||1997|
|BBa_K2201202||Composite||Tyrosyl tRNA/aminoacyl-synthetase for the incorporation of p-acetophenylalanine||Svenja Vinke||1558|
|BBa_K2201203||Composite||Tyrosyl tRNA/aminoacyl-synthetase for the incorporation of p-acetophenylalanine in response to CUA||Svenja Vinke||1558|
|BBa_K2201204||Composite||Tyrosyl tRNA/aminoacylsynthetase for the incorporation of L-(7-hydroxycoumarin-4-yl)ethylglycine||Svenja Vinke||1558|
|BBa_K2201343||Composite||Fusion protein of CFP and YFP with an amber codon in the linker under T7 promoter control||Svenja Vinke||1483|
|BBa_K2201024||Composite||Left flanking sequence + Terminator + repressing lacO||Lennard Karsten||1248|
|BBa_K2201025||Composite||Terminator + right flanking sequence||Lennard Karsten||1248|
|BBa_K2201028||Composite||Repair tempplate for HDR-mediated integration of cas9 into E. coli BL21 (DE3)||Lennard Karsten||3487|
|BBa_K2201032||Composite||sgRNA MAX target + mRFP-sacB fusion protein||Lennard Karsten||3487|
|BBa_K220140||Composite||sgRNA R-target mutA||Markus Haak||231|
|BBa_K2201041||Composite||sgRNA mutG||Markus Haak||231|
|BBa_K2201042||Composite||sgRNA R-target mutΔ||Markus Haak||231|
|BBa_K2201043||Composite||sgRNA R-target mutT||Markus Haak||231|
|BBa_K2201044||Composite||sgRNA R-target mutC||Markus Haak||231|
|BBa_K2201045||Composite||sgRNA MAX-target mutA||Markus Haak||256|
|BBa_K2201046||Composite||sgRNA MAX-target mutG||Markus Haak||256|
|BBa_K2201047||Composite||sgRNA MAX-target mutΔ||Markus Haak||256|
|BBa_K2201048||Composite||sgRNA MAX-target mutT||Markus Haak||256|
|BBa_K2201049||Composite||sgRNA MAX-target mutC||Markus Haak||256|
|BBa_K2201070||Composite||sgRNAs R-target mutA,G||Markus Haak||470|
|BBa_K2201071||Composite||sgRNAs R-target mutT,C||Markus Haak||470|
|BBa_K2201072||Composite||sgRNAs R-target A,G,Δ||Markus Haak||709|
|BBa_K2201073||Composite||sgRNAs MAX-target mutA,G,Δ,T,C||Markus Haak||1187|
|BBa_K2201074||Composite||sgRNAs MAX-target mutA,G||Markus Haak||520|
|BBa_K2201075||Composite||sgRNAs MAX-target mutT,C||Markus Haak||520|
|BBa_K2201076||Composite||sgRNAs MAX-target mutA,G,Δ||Markus Haak||784|
|BBa_K2201077||Composite||sgRNAs MAX-target mutA,G,Δ,T,C||Markus Haak||1312|
|BBa_K2201208||Composite||Cysteinyl-lysinyl-tRNA Synthetase/tRNA pair||Daniel Bergen||1860|
|BBa_K2201243||Composite||Fusion protein of CFP and YFP with an amber codon in the linker||Svenja Vinke||1431|
|BBa_K2201250||Composite||elastine consensus for PRe-RDL||Daniel Bergen||52|
|BBa_K2201251||Composite||silk consensus for PRe-RDL||Daniel Bergen||55|
|BBa_K2201320||Composite||Functional GFP-streptavidin fusion protein with medium gly-gly-ser linker and T7-promoter and RBS||Yannic Kerkhoff||1169|
|BBa_K2201321||Composite||NM-domain of Sup35 with an amber codon at position 21 under T7 promoter control||Svenja Vinke||837|
|BBa_K2201342||Composite||Fusion protein of RFP and GFP with an amber codon in the linker under T7 promoter control||Svenja Vinke||1489|
|BBa_K2201360||Composite||Our part BBa_K2201260||Maximilian Edich||7649|
|BBa_K2201361||Composite||Our part BBa_K2201261 after the Carboxysome and T7 promoter||Maximilian Edich||6946|
|BBa_K2201362||Composite||Our part BBa_K2201262 after the carboxysome and T7 promoter||Maximilian Edich||6946|
|BBa_K2201363||Composite||Our part BBa_K2201263 after the carboxysome and T7 promoter||Maximilian Edich||6946|
|BBa_K2201364||Composite||Our part BBa_K2201264||Maximilian Edich||6946|
|BBa_K2201365||Composite||Our part BBa_K2201265 after the Carboxysome and the T7 promoter||Maximilian Edich||6946|
|BBa_K2201366||Composite||Our part BBa_K2201266 after the Carboxysome and the T7 promoter||Maximilian Edich||6946|
|BBa_K2201367||Composite||Our part BBa_K2201267 after the Carboxysome and the T7 promoter||Maximilian Edich||6955|
|BBa_K2201368||Composite||Our part BBa_K2201268 after the Carboxysome and the T7 promoter||Maximilian Edich||6937|
|BBa_K2201404||Composite||Reverse Sequence of I746909||Olga Schmidt||924|
|BBa_K2201900||Composite||Positive selection plasmid||Svenja Vinke||1158|
|BBa_K2201901||Composite||Negative selection plasmid||Svenja Vinke||681|
McGraw, N. J., Bailey, J. N., Cleaves, G. R., Dembinski, D. R., Gocke, C. R., Joliffe, L. K., MacWright, R. S.&McAllister, W. T. (1985) Nucleic Acids Res. 13, 6753–6766.