Team:Caltech/TXTL

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TXTL Promoter Characterization

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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-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 figure shows the in vitro RFP fluorescence values measured for the same promoters. For ease of comparison, the x-axes on both 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 activity of weaker constitutive promoters tended to have more variation with respect to what was observed in vivo, but because RFP fluorescence was relatively low for these promoters (the lowest RFP fluorescence values were on par with values observed for the negative control), the variations may be a result of noise in the plate reader measurements. Besides that, the only significant discrepancies in relative promoter strengths came from parts J23107 and J23118. This suggests that the Anderson constitutive promoter family generally maintains the same relative strengths, as reported by Berkeley iGEM 2006, in TX-TL.

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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 <b>References</b>
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 [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.
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 [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).
+<br>
 [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.