Team:Caltech/TXTL

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TX-TL Characterization of Anderson promoters

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 figure shows the RFP fluorescence values (normalized by the maximum) measured in vivo by Anderson et al for different constitutive promoter constructs. Along with in vivo fluorescence values, the figure also includes the normalized in vitro RFP fluorescence values measured for the same promoters.

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. Despite some inconsistencies here and there, it is evident that the weakest promoters in vivo are also the weakest in TX-TL, and the strongest promoters in vivo are also the strongest in TX-TL. Overall, the data seem to show that the Anderson family of constitutive promoters behave essentially the same in vivo as they do in vitro using 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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-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 <i>in vivo</i> by Anderson <i>et al</i> for different constitutive promoter constructs. The second two figure show the <i>in vitro</i> 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. <br><br>
+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 figure shows the RFP fluorescence values (normalized by the maximum) measured <i>in vivo</i> by Anderson <i>et al</i> for different constitutive promoter constructs. Along with <i>in vivo</i> fluorescence values, the figure also includes the normalized <i>in vitro</i> RFP fluorescence values measured for the same promoters. <br><br>
+<img src="https://static.igem.org/mediawiki/2014/1/18/Caltech_2014_08_07_TXTL_promoter_overlaid.png" width="1000px">
-<img src="https://static.igem.org/mediawiki/2014/1/1b/BerkiGEM2006_promoter.png" width="1000px">
-<img src="https://static.igem.org/mediawiki/2014/3/30/TXTL_promoter_charact.png" width="1000px">
 <br><br>
-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 <i>in vivo</i>. The second TX-TL trial run seemed to differ more from the <i>in vivo</i> results than did the first trial run, but in general, the promoters that had strong expression levels <i>in vivo</i> were also strong <i>in vitro</i>, and the promoters that had weak expression levels <i>in vivo</i> were weak <i>in vitro</i>. Although the relative promoter strengths from the first TX-TL trial run seem to match well with what was observed <i>in vivo</i>, the second trial run indicates that the relative strengths are <i>not</i> necessarily the same <i>in vitro</i> as they are <i>in vivo</i>.
+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 <i>in vivo</i>. Despite some inconsistencies here and there, it is evident that the weakest promoters <i>in vivo</i> are also the weakest in TX-TL, and the strongest promoters <i>in vivo</i> are also the strongest in TX-TL. Overall, the data seem to show that the Anderson family of constitutive promoters behave essentially the same <i>in vivo</i> as they do <i>in vitro</i> using TX-TL.
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