Difference between revisions of "Part:BBa K2680550"
(→Results) |
(→Results) |
||
Line 35: | Line 35: | ||
Group1: Pre-expression for an hour | Group1: Pre-expression for an hour | ||
<html> | <html> | ||
+ | <br> | ||
<img src="https://static.igem.org/mediawiki/parts/8/8c/T--ZJUT-China--pre_expression_1.png" width="400" style="margin-top: 50px;"> | <img src="https://static.igem.org/mediawiki/parts/8/8c/T--ZJUT-China--pre_expression_1.png" width="400" style="margin-top: 50px;"> | ||
+ | <br> | ||
</html> | </html> | ||
Group2:Co-expression | Group2:Co-expression |
Revision as of 01:08, 11 October 2021
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23INCOMPATIBLE WITH RFC[23]Unknown
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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 cell-free system, protein degradation by clpXP is described by a zeroth order chemical kinetic,and clpxp protein can recognize the protein with ssrA tag, so it is useful to add clpxp degrading the degfp. This year,on the basis work of iGEM18_William_and_Mary, ZJUT-China measured the different eGFP degradation rate by adding different concentrate of plasmid P70-clpxp (Part:BBa_K3885203) based on the reference. We hope it will support more help on modelling and further 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
Group1: Pre-expression for an hour
Group2:Co-expression
ClpXP [nM] | eGFP degradation rate [nM/min] |
0 | 9.3 |
0.2 | 29.4 |
0.4 | 47.3 |
1 | 80.1 |
2 | 128.9 |
6 | 128.4 |
Degradation rate of deGFP upon different expression methods of clpXP. The degradation rate of deGFP-ssra only using the endogenous clpXP has been determined by an assay, which is achieved by measuring the kinetics after adding pure His-GFP-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.