Part:BBa_I723017
XylR coding region
The protein encoded by XylR recognises the chemical xylene (a petroleum derivative commonly found as a pollutant near industrial sites) and undergoes a conformational change allowing it to bind to, and positively regulate, the promoter Pu. XylR, therefore, should be used in combination with Pu to achieve a switch that activates in response to xylene.
Usage and Biology
Also responds to toluene and benzene (see [http://2007.igem.org/Glasgow/Wetlab/Results Glasgow 2007 wiki]).
In 2015 the Consort_Alberta removed the Pst cut site and used this part in the creation of the composite part: ECOS---> https://parts.igem.org/Part:BBa_K1834847
For the XylR gene with the Pstl cut site removed see: https://parts.igem.org/Part:BBa_K1834844
The ECOS BioBrick:
The following is work done by the Consort_Alberta 2015 team:
The resulting BioBrick consists of two main portions. The first portion of this plasmid is responsible for producing the protein XylR. This is essential because the bonding of m-xylene undergoes a conformational change when bonded to XylR which then allows it to bind to, and positively regulate, the Pu promoter. At a 4:2 XylR to Xylene ratio the bonding starts the production of our reporter protein. The second portion of our plasmid expresses the indicator protein when in the presence of bonded XylR and m-xylene and essentially allow us to know if m-xylene is present in our soil sample or not. We have had ECOS synthesized by GenScript from pre-existing parts in the registry. ECOS consists of the following parts:
We also attached the Reporter AmilCP in the backbone pCB1C3 to give us an output in correspondence to the level of xylene present. This was key to our lab work to ensure that ECOS worked properly and sucessfully detected xylene.
The Results:
We were able to successfully create two parts. The first being just ECOS and the second having AmilCP attached. We did a lab on the part including AmilCP and did have positive results! We are seeing production of AmilCP in the presence of xylene and in correlation to the concentration of xylene. During the second trial we used different concentrations of xylene to add to our 10mL of LB. We found that this time, leaving them in the incubator for shorter periods of time, we had much more uniform growth. Our output therefore did not have any correlation with the amount of cell growth. We did see very positive results. We had the darkest cells in the 50 uL tube and no dark cells in the negative control. We also were able to detect 50ppm based on volume. We grew cells in 10mL of LB and added different concentrations of xylene to the tubes. The lowest concentration we were able to see results with was .5 microlitres. Or 50ppm based on volume. We attempted to do trials with plates but they simply overgrew and gave us invalid results. We also did a trial with our prototype using a soil sample containing xylene. We detected 100ppm by volume with the prototype. We mixed 350mL of packed soil with 350uL of xylene, then we placed the soil in the upper chamber, making sure we didn't contaminate the sample with our hands/gloves. Then we placed our ECOS in the lower chamber and secured the entire unit to the shaker table and turning on our double fan system to circulate the air between the xylene in the soil sample and the ECOS in the lower chamber. We left it for 13 hours (4 p.m. to 10 a.m. the next morning).
For more details please view our Lab Write up https://static.igem.org/mediawiki/2015/3/3c/Consort-iGem-Lab-Write-Up.pdf and our project page: http://2015.igem.org/Team:Consort_Alberta/project
As Bilkent_UNAMBG 2016 team, we did codon optimisation for E. Coli of this part from Uniprot amino acid sequence. see: https://parts.igem.org/Part:BBa_K1946005
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 643
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 466
Illegal PstI site found at 643 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 86
Illegal BglII site found at 1548 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 643
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 643
Illegal NgoMIV site found at 252 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 739
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