Difference between revisions of "Part:BBa K3093002"
Still Water (Talk | contribs) |
Still Water (Talk | contribs) |
||
| Line 5: | Line 5: | ||
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. | 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. | ||
| − | <html><img style="width:600px;padding-left:100px;" src="https://2019.igem.org/wiki/images/4/41/T--ECUST_China--regulator_circuit-cello.png"> </html> | + | <html><img style="width:600px;padding-left:100px;" src="https://2019.igem.org/wiki/images/4/41/T--ECUST_China--regulator_circuit-cello.png"> </html><br><span style="font-size: 14px;"> |
<b>Figure 3.7</b> Gene circuit of cellobiose response element</span> | <b>Figure 3.7</b> Gene circuit of cellobiose response element</span> | ||
| Line 12: | Line 12: | ||
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 : chbRN238S, chbRY30C and chbRN137K. | 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 : chbRN238S, chbRY30C and chbRN137K. | ||
| − | <html><img style="width:600px;padding-left:100px;" src="https://2019.igem.org/wiki/images/b/bb/T--ECUST_China--regulator_cellomrfp.png"> </html> | + | <html><img style="width:600px;padding-left:100px;" src="https://2019.igem.org/wiki/images/b/bb/T--ECUST_China--regulator_cellomrfp.png"> </html><br><span style="font-size: 14px;"> |
<b>Figure 3.10</b> The PCR results of pCEL backbone and chbR</span> | <b>Figure 3.10</b> The PCR results of pCEL backbone and chbR</span> | ||
Revision as of 13:55, 20 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.
Figure 3.7 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.
During the experiment, we used mRFP as the reporter. The sequence of Pcel 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 : chbRN238S, chbRY30C and chbRN137K.
Figure 3.10 The PCR results of pCEL backbone and chbR
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.
Figure 3.11 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 10 mM cellobiose whereas no induction was seen in the presence of wild-type ChbR.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]