Team:Caltech/TXTL

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<tr><td align=center valign=center bgColor=#000000> <font size=+2 color=#FFFFFF> Introduction to TXTL </font> </td></tr>
 
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What is TXTL
 
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<tr><td bgColor=#FFFFFF colspan = 3 height = 100px> <font size=+4> <center> TX-TL Characterization of Anderson promoters </center></font></td> </tr>
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<tr><td bgColor=#000000 align=center> <font size=+2 color="#FFFFFF"> Anderson Promoters </font> </td></tr>
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Characterized family of constitutive promoters in TXTL <br>
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<p>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 <i>in vitro</i> process, behavior of components such as promoters, ribosome binding sites, and terminators may behave differently than <i>in vivo</i>. Because of this discrepancy, it is necessary to characterize different promoter strengths in TX-TL.  </p>
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<table width=70%><tr><td><b>Figure 1. Use of TX-TL as a breadboard for rapid prototyping and debugging of circuits.</b>  
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<p>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. </p>
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<tr><td > <h3> iGEM Team attributions page</h3></td>
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<table width=70%><tr><td><b>Figure 2. Normalized RFP fluorescence under different constitutive promoters, both <i>in vivo</i> and in TX-TL.</b>
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Each team must clearly attribute work done by the student team members on this page. The team must distinguish work done by the students from work done by others, including the host labs, advisors, instructors, and individuals not on the team roster.
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<p>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. </p>
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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).
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We have this requirement to help the judges know what you did yourselves and what you had help with. We don't mind if you get help with difficult or complex techniques, just be sure to report the work your team did and the work that was done by others.  
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For example, you might choose to work with an animal model during your project. Working with animals requires getting a license and applying far in advance to conduct certain experiments in many countries. This is something that is difficult to achieve during the course of a summer, but much easier if you can work with a postdoc or PI who has the right licenses.
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[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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A great example of complete attribution comes from the <a href="https://2011.igem.org/Team:Imperial_College_London/Team">Imperial College London 2011 team</a> (scroll down to the bottom of their team page to see attributions).  
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Here are some of the fields we recommend you have on this page. If there are other areas not listed below, but applicable to your team/project, please feel free to also list them on your attributions page. Please feel free to remove any areas not applicable to your project.
 
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<li>General Support</li>
 
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<li> Thanks and acknowledgements for all other people involved in helping make a successful iGEM team.</li>
 
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Latest revision as of 04:57, 16 October 2014


Home Team Official Team Profile Project Parts TXTL Promoter Characterization Notebook Safety Attributions
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.



Figure 1. Use of TX-TL as a breadboard for rapid prototyping and debugging of circuits.


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.



Figure 2. Normalized RFP fluorescence under different constitutive promoters, both in vivo and in TX-TL.


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.