View source for Part:BBa K2333411


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<partinfo>BBa_K2333411 short</partinfo>

This part is designed to facilitate easy measurement of the strength of protein degradation tag (pdt) E by measuring time to steady-state fluorescence values of sfGFP under the control of the strong constitutive promoter <partinfo>Bba_J23100</partinfo>. William and Mary iGEM 2017 used pdts as a method to control gene expression speed.This part was utilized to characterize the degradation properties of pdt E and confirm the fact that different pdts have different degradation strengths. See [http://2017.igem.org/Team:William_and_Mary/Results William and Mary's 2017 project] for more details.
This part is one of a series of sfGFP reporter pdt parts. Series range is from <partinfo>BBa_K2333407</partinfo> to <partinfo>Bba_K2333412</partinfo>.


===Usage and Biology===
Protein degradation tag E (<partinfo>BBa_K2333005</partinfo>) is the second weakest of the 6 protein degradation tags that William and Mary 2017 characterized, and is associated with the E. Coli orthogonal protease mf-Lon (<partinfo>Bba_K2333011</partinfo>). Therefore this part has the second smallest degradation rate of the 6 protein degradation tags and it is the second slowest to reach the steady-state fluorescence value. This part contains J23100 constitutive promoter, <partinfo>Bba_B0034</partinfo> (RBS),pdt E, a double stop codon and <partinfo>Bba_B0015</partinfo> (double terminator) in the William and Mary iGEM Universal Nucleotide Sequences (UNS) format. See Torella, et. al (2013). In order to demonstrate that protein degradation tags operated similarily regardless of the tagged protein, sfGFP reporters that were analogous to the mScarlet-I parts (<partinfo>Bba_K2333413</partinfo> to <partinfo>Bba_K2333419</partinfo>) were built and characterized. This demonstrates that the protein degradation tags are modular and that they have differential strengths even when they are tagged on different proteins.

===Characterization===
W&M 2017 preliminarily characterized this Pdt E tagged <partinfo>Bba_J23100</partinfo> sfGFP construct along with IPTG-inducible mf-Lon protease to show degradation rate and speed change effect measurements. The graph below shows this degradation characterization along with the data from the other tags in this series (<partinfo>K2333407</partinfo>-<partinfo>K2333412</partinfo>).While degradation is occurring, it does not line up with the expected tag strengths correctly. The deviation from expected strengths is possibly due to the reporter sfGFP that was used or due to the fact that these measurements were only performed in a plate reader due to time constraints. 

<html><img src="https://static.igem.org/mediawiki/parts/7/74/T--William_and_Mary--J23100_sfGFP.png" width="520px"/></html>

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K2333411 SequenceAndFeatures</partinfo>

===References===
[1] Torella JP, Boehm CR, Lienert F, Chen J-H, Way JC, Silver PA. Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic Acids Research. 2013;42(1):681–689.

[2] Cameron DE, Collins JJ. Tunable protein degradation in bacteria. Nature Biotechnology. 2014;32(12):1276–1281. 

[3] Lou, C., Stanton, B., Chen, Y.-J., Munsky, B., & Voigt, C. A. (2012). Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology, 30(11), 1137–1142. http://doi.org/10.1038/nbt.2401


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===Functional Parameters===
<partinfo>BBa_K2333411 parameters</partinfo>
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