Difference between revisions of "Part:BBa K2333410"

Revision as of 19:12, 1 November 2017

UNS J23100 sfGFP pdt D

This part is designed to facilitate easy measurement of the strength of protein degradation tag (pdt) D by measuring time to steady-state fluorescence values of sfGFP under the control of the strong constitutive promoter BBa_J23100. 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 D 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 BBa_K2333407 to BBa_K2333412

Usage and Biology

Protein degradation tag D (BBa_K2333004) is of medium strength among the 6 protein degradation tags that William and Mary 2017 characterized, and is associated with the E. Coli orthogonal protease mf-Lon (BBa_K2333011). Therefore this part has a medium degradation rate among the 6 protein degradation tags and it is the third slowest to reach the steady-state fluorescence value. This part contains J23100 constitutive promoter, BBa_B0034 (RBS),pdt D, a double stop codon and BBa_B0015 (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 (BBa_K2333413 to BBa_K2333419) 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 A tagged BBa_J23100 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 (BBa_K2333407-BBa_K2333412).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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 47
Illegal NheI site found at 70
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 106

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

@@ Line 8: / Line 8: @@
 ===Usage and Biology===
 Protein degradation tag D (<partinfo>Bba_K2333004 </partinfo>) is of medium strength among 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 a medium degradation rate among the 6 protein degradation tags and it is the third slowest to reach the steady-state fluorescence value. This part contains J23100 constitutive promoter, <partinfo>Bba_B0034</partinfo> (RBS),pdt D, 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 A 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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