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Team:Caltech/TXTL
From 2014.igem.org
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| TX-TL is a cell-free transcription translation system that allows for inexpensive and rapid prototyping of biological circuits. This is desirable as the current method of prototyping and debugging circuits requires DNA parts to be cloned into cells, which can take a long time. With TX-TL, once all the DNA parts have been obtained, the circuit can be tested immediately, and so several circuit iterations can be tested in the time it takes to successfully clone even one circuit iteration into cells. Since TX-TL is an in vitro process, behavior of components such as promoters, ribosome binding sites, and terminators may behave differently than in vivo. Because of this discrepancy, it is necessary to characterize different promoter strengths in TX-TL. | |||||||||||||
We chose to characterize the strength of the Anderson family of constitutive promoters (Berkeley iGEM 2006) in TX-TL. We used biobrick parts J23100 through J23118, with the exception of parts J23108, J23109, and J23111. The first figure shows the reported RFP fluorescence values measured in vivo by Anderson et al for different constitutive promoter constructs. The second two figure show the in vitro RFP fluorescence values measured for the same promoters, for two different trials. For ease of comparison, the x-axes on all three figures are the same. 
As is evident from the data, the overall trend of relative promoter strengths in TX-TL seems to be fairly consistent with what was observed in vivo. The second TX-TL trial run seemed to differ more from the in vivo results than did the first trial run, but in general, the promoters that had strong expression levels in vivo were also strong in vitro, and the promoters that had weak expression levels in vivo were weak in vitro. Although the relative promoter strengths from the first TX-TL trial run seem to match well with what was observed in vivo, the second trial run indicates that the relative strengths are not necessarily the same in vitro as they are in vivo. |
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References
[1] J. Shin and V. Noireaux, An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells. ACS Synthetic Biology, 1(1):29–41, 2012. [2] Z. Z. Sun, C. A. Hayes, J. Shin, F. Caschera, R. M. Murray, V. Noireaux, Protocols for Implementing an Escherichia Coli Based TX-TL Cell-Free Expression System for Synthetic Biology. Journal. of Visualized Experiments (JoVE), e50762, doi:10.3791/50762 (2013). [3] Z. Z. Sun, E. Yeung, C. A. Hayes, V. Noireaux and Richard M. Murray, Linear DNA for rapid prototyping of synthetic biological circuits in an Escherichia coli based TX-TL cell-free system. ACS Synthetic Biology, 2014. |
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