Stanford/BIOE44:Module3:Day2:nitrate

STATUS
Loaded up at DNA2.0 and authors have confirmed sequence is correct. Good to order!

Nitrate Sensor Project

Authors: Scott Runyon and Travis Urban

Novel Bio Bricks Part: Bba_NRL001

Long Description: The NarL protein is a cytoplasmic transcription factor that serves as the second component in the two-component Nar pathway found in E. Coli K-12. When NarX (a membrane protein) binds nitrate, it autophosphorylates and then acts as a histidine kinase on the NarL protein. When NarL is phosphorylated, it binds bacterial DNA and acts as a repressor/activator, depending on the particular operon.

Short Description: Coding sequence for the NarL protein, a response regulator in Nar pathway, in accordance with BioBrick assembly 10.

Sequence: gaattcgcggccgcttctagATGAGTAATCAGGAACCGGCTACTATCCTGCTGATTGACGATCACCCGATGCTGCGAACTGGCGTAAAACAGCTTATCAGTATGGCACCAGATATCACCGTGGTTGGCGAAGCGAGTAATGGCGAACAGGGTATTGAACTGGCGGAGTCTCTTGATCCCGATCTGATCCTGTTAGATCTCAATATGCCCGGCATGAACGGTCTGGAAACGCTGGATAAACTGCGCGAAAAGTCCCTCTCAGGGCGCATTGTGGTATTCAGCGTCTCTAACCATGAAGAAGATGTGGTCACCGCACTGAAACGCGGCGCGGATGGCTATCTGTTAAAAGATATGGAACCGGAAGATCTGCTGAAAGCATTGCATCAGGCAGCTGCTGGCGAAATGGTATTAAGCGAAGCATTAACGCCTGTTCTGGCCGCCAGCTTGCGCGCTAACCGTGCCACTACTGAGCGCGATGTTAACCAGTTAACCCCACGCGAGCGCGATATTCTCAAGCTGATTGCCCAGGGTTTGCCGAACAAGATGATTGCCCGCCGCCTGGATATCACCGAAAGCACAGTAAAAGTGCACGTCAAGCACATGCTGAAGAAAATGAAGCTCAAGTCTCGCGTGGAAGCAGCGGTATGGGTGCATCAGGAGCGCATTTTCTAAtactagtagcggccgctgcag

Natural NarL sequence did not contain any EcoRI, PstI, SpeI, or XbaI restriction sites. NarL part is therefore usable by BioBrick Assmebly 10 standards.

References:

Stewart, V., H. Lin, P. Bledsoe. 2007. Activation of yeaR-yoaG Operon Transcription by the Nitrate-Responsive Regulator NarL Is Independent of Oxygen-Responsive Regulator Fnr in Escherichia coli K-12. J. Bacteriol. 189: 7539 - 7548

Stewart, V., R. S. Rabin 1993. Dual Response Regulators (NarL and NarP) Interact with Dual Sensors (NarX and NarQ) To Control Nitrate - and Nitrite - Regulated Gene Expression in Escherichia coli K-12. J. Bacteriol. 175: 3259 - 3268

Sequence Information

Link to NarL Sequence Information

Additional Resources

after downloading the Ape sequence recognition program, http://www.biology.utah.edu/jorgensen/wayned/ape/ we checked the 650 bp sequence for EcoRI, Xba1, SpeI, and Pst1 restriction sites (none) which we send out to be synthesized.

Validated that the part we ordered to be synthesized and the part we designed were the same, http://workbench.sdsc.edu/

primers were developed from the following program (which adds biobrick parts), http://ginkgobioworks.com/cgi/primer.cgi