Difference between revisions of "Part:BBa K3071004"

Revision as of 20:42, 18 October 2019

C reactive protein-like protein (Clp) from Xanthomonas campestris pv. Campestris

Description

This the protein-encoding gene for the C reactive protein-like protein (Clp) from Xanthomonas campestris pv. Campestris, which is codon-optimized to express in escherica coli K12 strain. This part is developed and improved based on the previous recorded part (BBa K1315005). The SDS-PAGE gel data and western blot data of our composite part (BBa_K3071020) shows it could be correctly expressed in E. coli in form of fusion protein.

Biology

C reactive protein-like protein (Clp) is a global transcriptional regulator that regulates virulence factors production by activating or repressing the expression of a large set of genes in the diffusible signal factor DSF pathway. It also regulates the genes that involve in extracellular polysaccharide (EPS) synthesis, flagellum synthesis, protein, and fatty acid metabolism, multidrug resistance, iron uptake. They also regulate genes that encoding extracellular enzymes, membrane components, and a few transcription factors.

Usage

Clp is a DNA binding protein that binds effectively to the target promoter for gene expression. It upregulates the promoter by binding to CBS-I (BBa_K3071011) and CBS-II (BBa_K3071012) through a high GC central region. Its function can be affected by cyclic-di-GMP that binds strongly on clp. The glutamic acid residue (E99) in the cNMP binding domain of the clp contributes to the high affinity between clp and cyclic-di-GMP. Once the clp is bound by cyclic-di-GMP, the conformation of clp is changed allosterically. Thus, the interaction between clp and the promoter is hindered and the gene expression is suppressed.

Characterization

Figure 1 Western blot analysis of Clp protein expression using Myc-Tag (9B11) Mouse mAb (1:2000)

Clone were induced by 1mM IPTG at 32℃ and collected at different time points, which are 12hr, 16hr, 20hr, 24hr, and 28hr. After blotting with corresponding antibodies, Clp-PspF was confirmed with successful expression at all time points. Results of blots probing Clp-pspF showed that clones collected at 12hr contained the highest amount of target proteins and the protein quantity decreased from 16hr to 28hr. This may due to degradation inside the cells.

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
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 70
1000
COMPATIBLE WITH RFC[1000]

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 <center><u>Figure 1  Western blot analysis of Clp protein expression using Myc-Tag (9B11) Mouse mAb (1:2000)</u></center>
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-Clones were induced by 1mM IPTG at 32℃  and collected at different time points, which are 12hr, 16hr, 20hr, 24hr, and 28hr. After blotting with corresponding antibodies, Clp-PspF was confirmed with successful expression at all time points. Results of blots probing Clp-pspF showed that clones collected at 12hr contained the highest amount of target proteins and the protein quantity decreased from 16hr to 28hr. This may due to degradation inside the cells.
+Clone were induced by 1mM IPTG at 32℃  and collected at different time points, which are 12hr, 16hr, 20hr, 24hr, and 28hr. After blotting with corresponding antibodies, Clp-PspF was confirmed with successful expression at all time points. Results of blots probing Clp-pspF showed that clones collected at 12hr contained the highest amount of target proteins and the protein quantity decreased from 16hr to 28hr. This may due to degradation inside the cells.
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