Difference between revisions of "Part:BBa K3093002"

Revision as of 00:16, 22 October 2019

Cellobiose operon

The cellobiose response element is our new biobrick. We chose the cellobiose as the response element ranther than glucose because cellobiose could accumulate in the cytosol, while glucose is easy to metabolize. Only when the cellobiose accumulated to a certain concentration, could the cellobiose operon be active to reverse the inverter.

Design

Figure 1. Gene circuit of cellobiose response element

Cellobiose response element is the mutant of chb operon in E.coli. Wild‐type strains of Escherichia coli are normally unable to metabolize cellobiose. The chb operon is the inducible genetic system involved in the catabolism of N,N′‐diacetylchitobiose. However, single base‐pair changes in the transcriptional regulator chbR that translate into single‐amino‐acid substitutions constitute the cellobiose operon which can response cellobiose. So, ECUST_China iGEMers performed several types of mutation: chbRN137K, chbRY30C and chbRN238S, aiming to find a efficient cellobiose response element.

Characterization

During the experiment, we used mRFP as the reporter. 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 : chbRN137K, chbRN238S and chbRY30C.

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.

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]

@@ Line 22: / Line 22: @@
 During the experiment, we used mRFP as the reporter. The sequence of <i>P<sub>cel</sub></i> and <i>chbR</i> was obtained from <i>E.coli</i> K12 MG1655 via PCR .Then we performed inverse PCR to achieve the site-directed mutation of amino acids of <i>chbR</i>. According to the literature, we chose three site-directed mutation : chbRN137K, chbRN238S and chbRY30C.
-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 10 mM cellobiose for about 40 hours. All of the samples were transferred to 96-well plate to measure the mRFP fluorescence.
+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.
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