Part:BBa_K2680550

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 左边表格，右边图片 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.