Part:BBa_K1706007
T7 RNA Polymerase gene
T7 RNA Polymerase is an RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA in the 5'→ 3' direction. T7 polymerase is extremely promoter-specific and transcribes only DNA downstream of a T7 promoter. The T7 polymerase also requires a DNA template and Mg2+ ion as cofactor for the synthesis of RNA. It has a very low error rate. T7 polymerase has a molecular weight of 99 kDa. In biotechnology applications, T7 RNA polymerase is commonly used to transcribe DNA that has been cloned into vectors that have two (different) phage promoters (e.g., T7 and T3, or T7 and SP6) in opposite orientation. RNA can be selectively synthesized from either strand of the insert DNA with the different polymerases. The enzyme is stimulated by BSA or spermidine. Homogeneously labeled single-stranded RNA can be generated with this system. Transcripts can be non-radioactively labeled to high specific activity with certain labeled nucleotides.
The Influence of T7 RNA Polymerase on Phage Formation in Cell-Free Transcription-Translation Systems
The project of ETH Zürich iGEM team 2019 is focused on creating bacteriophage libraries with novel host specificities. Cell-free translation transcription systems allow for the high-yield production of bacteriophages in vitro. T7 RNA Polymerase has been a crucial component throughout our iGEM project as the bacteriophage T7 is our declared main player in the experimental set-up. The Arbor linear myTXTL – a cell free transcription translation system - has been used for the in vitro formation of phages from the T7 genome. myTXTL uses a sigma70 factor for the transcription of template DNA. If it is desired to initiate transcription by a T7 promotor it is crucial to add the T7 RNAP to the reaction mixture. This is achieved by a plasmid where the expression of T7 RNA Polymerase is driven by a P70a promotor that is recognized by the sigma70 RNA polymerase present in the myTXTL master mix. A detailed description of the following experimental setup can be found here.
The T7 genome encodes the T7 RNA Polymerase. It’s expression is driven by a promotor that is recognized by the sigma70 RNA polymerase. Therefore, it is not essential to add additional T7 RNA Polymerase to the myTXTL reaction for the successful in vitro formation of T7 phages. However, as we want to optimize the formation of phages in cell free translation transcription system, we wanted to test whether additional T7 RNAP would boost the formation of phages.
Literature [1] provides evidence that increasing template DNA concentrations leads to higher protein yields in myTXTL reactions. A control experiment with varying concentrations of a plasmid containing deGFP driven by a T7 promotor verifies that increasing the DNA template concentration of deGFP leads to higher protein yields (figure 1A). Note that due to the T7 promotor, T7 RNAP has been added to the reaction mixture (0.1 nM), but its concentration has been kept constant. In a second experiment, the concentration of T7 RNAP has been varied while keeping the deGFP concentration constant (figure 1B). The measured deGFP concentration shows a small increase when T7 RNAP plasmid concentration was increased from 0.001 nM to 0.1 nM. Interestingly, adding an excess of 2.8 nM T7 RNAP leads to a significant decrease in deGFP yield. This can be explained by the fact that the formation of T7 RNAP will use up some of the energy in the reaction mixture and will therefore lead to a decrease in the available energy for deGFP production.
The main question of this experiment was whether the general principle of forming more protein by adding more RNAP plasmid to the reaction mixture would also lead to a higher formation of phages. The myTXTL reaction has been performed according to our phage formation protocol (link). This protocol adds dNTPs, PEG800 and T7 DNA to the myTXTL mixture. These values have been kept constant.
The number of formed phages has been determined by performing spot assays followed by plaque assays for more precise quantification. These assays are used to demonstrated the infection capacity of the phages for the E.coli strain DH5a, which allows a conclusion of how successful the formation of T7 phages in myTXTL under varying RNA Polymerase concentrations was.
Figure 2 shows that increasing the T7 RNA Polymerase concentration in the myTXTL reaction mixture leads to a significant decrease in the formation of functional phages and is therefore not beneficial. An addition of zero RNA polymerase does nevertheless not mean that there was no RNA Polymerase present in the reaction volume, as the T7 DNA provides itself a gene for the expression of RNA Polymerase.
The results of the characterization show that the formation of phages cannot be simply seen as a connection of proteins. The T7 genome is organized into two parts that are driven by the bacterial and the T7 RNAP respectively (figure 3). The T7 RNA polymerase transcription rate is 5-10 faster than the one of an E. coli polymerase [2]. Therefore, the presence of T7 RNA polymerase in the reaction mixture will lead to the premature high-level transcription of T7 promotor driven genes and lead to an imbalance in bacteriophage protein composition.
Figure 3: The T7 genome is driven by two classes of promotors. The first part is driven by bacterial promotors which also drive the expression of the T7 RNA polymerase. The second part is driven by several T7 promotors. T7 RNA polymerase has 5-10 times faster transcription rates than E. coli RNA polymerases. If the T7 RNA polymerase is present too early, the fast transcription of genes driven by the T7 promotos will lead to an imbalance in the stoichiometry of the phage forming proteins.
To conclude, the addition of phage specific RNA polymerases into cell-free transcription translation systems is not recommended as it will interfere with the stoichiometry of the phage proteins.
Bibliography
[1] Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction, Synthetic Biology, Volume 3, Issue 1, 2018, ysy002
[2] Springman, R., Badgett, M. R., Molineux, I. J., & Bull, J. J. (2005). Gene order constrains adaptation in bacteriophage T7. Virology, 341(1), 141-152
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T7 RNA polymerase in synthetic biology
The T7 RNA polymerase is a single-subunit enzyme that can be used for orthogonal and specific expression in various hosts, since it has high specificity to its own promoter and only two types of terminators have been confirmed to work for T7 RNA polymerase, class I and class II [1].
The T7 RNAP with its high transcription rates causes a lot of stress to the host’s cells, so the T7 RNAP needs to be integrated into the host genome and theT7 promoter in a plasmid. Researchers have tried to place the T7 RNAP and T7 promoter together in a single plasmid but the stress caused the death of the host cells [1].
Bibliography
[1] Wang, W., Li, Y., Wang, Y., Shi, C., Li, C., Li, Q., & Linhardt, R. J. (2018). Bacteriophage T7 transcription system: an enabling tool in synthetic biology. Biotechnology Advances, 36(8), 2129–2137. doi:10.1016/j.biotechadv.2018.10.001
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