Difference between revisions of "Part:BBa K656013"

Latest revision as of 23:00, 28 October 2011

Sporosarcina pasteurii Urease

This genetic cassette represents the first modular, fully functional Sporosarcina pasteurii urease suite in the iGEM registry. It contains an 11kb gene cluster which encodes the multiple subunits and other genes necessary for the function of urease in Sporosarcina pasteurii. E. coli genetically transformed with this sequence has the ability to hydrolyze urea. Ureolytic activity of transformants has been confirmed using urease test plates, which change color as urea is hydrolyzed increases the pH of the medium. To see an image of urease plates changing color due to the activity of transoformants (streaked directly on the plate), please use the following link: http://2011.igem.org/File:Transformant_urease_plates.JPG.

The sequence of this cassette is currently unavailable because it currently does not exist in any known database, but we have begun to sequence it with the assistance of Dr. Chris Mason (Weill Cornell Medical College). As soon as the sequence data is available, we will supplement the directory listing.

Characterization As a qualitative assay of urease activity, we selected transformant colonies at random from the LB-chlor growth plates and grew them up in liquid LB-chlor culture. These monoclonal cultures were then plated and allowed to grow on urease test plates; if urease is present and active in the cultures, the urea is cleaved in the surrounding agar, causing the pH of the surrounding agar to increase. Phenol-red then indicates the subsequent increase in pH from ureolysis. All but one of the colonies we selected off the LB-Chlor plate exhibited ureolytic activity, indicating that our workflow had resulted in the construction of a functional S. pasteurii urease biobrick. From the urease test plates we were able to determine that our part did in fact, function, albeit qualititatively.

Urease test plates displaying ureolytic activity of recombinant E. coli expressing part BBa_K656013

To more accurately and quantitatively measure the activity of the urease biobrick, we hope to perform a conductivity assay protocol with our transformants.

The gel above depicts the urease cassette construct digested with EcoRI restriction enzyme

The gel above depicts the urease cassette construct digested with PstI restruction enzyme

Another component of our characterization of part BBa_K656013 was a determination of the construct's compatibility with the biobrick assembly method. Because we lack the sequence data for this part, we performed the basic biobrick restriction digests (EcoRI, XbaI ,SpeI , and PstI) on our urease construct to determine if there were any cut sites within the operon itself. Our construct did indeed contain multiple biobrick restriction sites, specifically EcoRI and PstI, both of which cut our operon into multiple pieces. Again, because of our lack of a sequence for this part, it is impossible to say for sure where in the operon these cut sites exist; fortunately, neither XbaI nor SpeI enzymes cut our operon into distinct pieces, allowing us to incorporate the construct into biobrick submission plasmid pSB1C3. Therefore, the part we created is compatible with standard biobrick assembly, but should not be used with the outer restriction enzymes EcoRI and PstI.

Documentation of the BioBrick part: http://2011.igem.org/Team:Brown-Stanford/REGObricks/Biobrick

For information on qualitative and quantitative characterization of this part's function: http://2011.igem.org/Team:Brown-Stanford/REGObricks/Characterization

Sequence and Features