Part:BBa_K3093000

chbR N238S Y30C

"Chitobiose regulator," ChbR, is a dual transcriptional factor that regulates an operon (chb) involved in transport and degradation of the -glucoside N,N'-diacetylchitobiose (chitobiose) . [More information is available at EcoCyc: EG10143].

Usage and Biology

ChbR is encoded within the chb operon itself. In the presence of chitobiose, ChbR binds to two direct repeats located between two NagC binding sites and activates the transcription of the chb operon by overlapping the -35 box of chbBp. In the absence of chitobiose, ChbR acts as a repressor of the chb operon. Monoacetylchitobiose-6-P has been experimentally determined to be the inducer for ChbR.

Construction

The sequence of P_cel and chbR was obtained from E.coli K12 MG1655 via PCR .Then we performed inverse PCR to achieve the site-directed mutation of amino acids of chbR. According to the literature, we chose three site-directed mutation : ChbR N238S;ChbR Y30C; ChbR N137K.

Figure 1. The site-directed mutation of ChbR N238S

Figure 2. The site-directed mutation of ChbR Y30C

Figure 3. The site-directed mutation of ChbR N137K

Characterization

During the experiment, we used mRFP as the reporter. After constructing the mutation plasmids: pIN1-NK and pIN1-YC-NS. Both of the mutant clones and the wild-type strain were incubated in M9 medium containing 0.4% glycerol and 0.4% casamino acids, with or without cellobiose for about 40 hours. All of the samples were transferred to 96-well plate to measure the mRFP fluorescence.

Figure 2. Fluorescence intensity induced by cellobiose

The results revealed that presence of both chbR mutants resulted in a high basal level of expression. More importantly, the transformant carrying ChbRYC-NS showed an approximately threefold induction. Over the basal level in the presence of cellobiose whereas no induction was seen in the presence of wild-type ChbR.

References

[1]Plumbridge J, Pellegrini O. Expression of the chitobiose operon of Escherichia coli is regulated by three transcription factors: NagC, ChbR and CAP. Mol Microbiol. 2004 Apr;52(2):437-49. PubMed PMID: 15066032.

[2]Kachroo AH, Kancherla AK, Singh NS, Varshney U, Mahadevan S. Mutations that alter the regulation of the chb operon of Escherichia coli allow utilization of cellobiose. Mol Microbiol. 2007 Dec;66(6):1382-95. Epub 2007 Nov 19. PubMed PMID: 18028317.

[3] Joseph AM, Sonowal R, Mahadevan S. A comparative study of the evolution of cellobiose utilization in Escherichia coli and Shigella sonnei. Arch Microbiol. 2017 Mar;199(2):247-257. doi: 10.1007/s00203-016-1299-0. Epub 2016 Oct 1. PubMed PMID: 27695910.

Sequence and Features

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
  COMPATIBLE WITH RFC[1000]