Part:BBa_K2680550

deGFP-ssra

de-GFP ssra is a destabilized GFP created by "fusing amino acids 422–461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP) ¹ This destabilized GFP is fitted with an ssrA degradation tag.

References

1. Li‡, X., Zhao, X., Fang, Y., Jiang, X., Duong, T., & Huang, C. F. (1998, December 25). Xianqiang Li. Retrieved from http://www.jbc.org/content/273/52/34970.full.html

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 47
  Illegal BsaI.rc site found at 771
  

Contribution by Team ZJUT-China 2021

Group: Team ZJUT-China 2021
Author: Lianjie Sha and Xia Yao
Summary: According to the lectures, we learned that the degradation rate of eGFP-ssrA could be measured. In the cell-free system, protein degradation by clpXP is described by a zeroth order chemical kinetic.In protein substrates (eGFP), ClpX recognizes ssrA--a specific C-terminal degradation tag, proceeds to unfold stable tertiary structure in the protein, and then spools or translocates the unfolded polypeptide chain into a sequestered proteolytic compartment in ClpP for degradation into small peptide fragments. As part of iGEM18_William_and_Mary, ZJUT-China assessed the degradation rate of different eGFP (Part:BBa_K2680550)by adding different concentrate of plasmid P70-ClpXP (Part:BBa_K3885203)based on the reference. We hope this will enhance further modeling and experiments.

Methodology

There are two methods to express clpxp protein: co-expression and pre-expression.Accelerated protein degradation can be achieved by co-expression of P70a-ClpXP, by adding protein to a cell-free system pre-incubated with P70a-ClpXP for an hour or by adding dilutions of pre-expressed ClpXP (P70a-ClpXP, 3nM). Different methods can provide different rates of protein degradation, ranging from 9.3 nM/min to 250 nM/min. By expressing ClpXP, protein synthesis can be adjusted to an appropriate rate[1].

Results

Figure 1 Degradation rate of deGFP upon different expression methods of ClpXP.

The degradation rate of eGFP-ssrA only using the endogenous ClpXP has been determined by an assay, which is achieved by measuring the kinetics after adding pure His-eGFP-ssrA (5μM) to the Cell-Free system.

Analysis

As shown in the figure above, the higher the concentration of ClpXP, the faster the degradation of eGFP-ssrA. Meanwhile, according to the table, when ClpXP with the same concentration was added, eGFP degradation rate in pre-expression was from 6.5 nM/min to 256 nM/min, while in co-expression, eGFP degradation rate was from 9.3 nM/min to 128 nM/min. It can be concluded that pre-expression is more conducive to eGFP protein degradation than co-expression.

Reference

[1] Garamella J, Marshall R, Rustad M, et al. The all E. coli TX-TL toolbox 2.0: a platform for cell-free synthetic biology[J]. ACS synthetic biology, 2016, 5(4): 344-355.