Part:BBa_M33002:Design
CDS for Fnr-LH28 protein, activates narGp in presence of nitrate.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The coding sequence was checked and edited for restriction enzyme sites to ensure that it is Assembly Standard 10-compliant. The sequence also contains a single missense mutation, converting Leucine 28 from the natural CDS to Histadine to allow expression of Fnr in aerobic conditions.
Afterward, the following edits were made: the last ten base-pairs before the CDS were deleted as they were part of the RBS (ribosome binding site) of the fnr (Shaw, Guest 1982). Also, the required Leucine to Histidine switch was made in the CDS. Finally, the sequence TAATAA replaced the last three nucleotides of the coding sequence.
Source
The part comes as a mutated form of the fumerate nitrate reductase coding sequence from the E. coli genome, a crucial part of the nitrate respiration cycle.
This part was directly copied from a genomic sequence--specifically from the EcoGene DNA database (ecogene.org). The selection was from 111 bp upstream of fnr CDS (coding sequence), to the end of the CDS.
References
1. Bates, D. M., C. V. Popescu, N. Khoroshilova, K. Vogt, H. Beinert, E. Munck, and P. J. Kiley."Substitution of Leucine 28 with Histidine in the Escherichia coli Transcription Factor FNR Results in Increased Stability of the [4Fe-4S]2+ Cluster to Oxygen" J. Biol. Chem. 2000 275: 6234-6240.
2. Bates, D. M., Lazazzera, B. A., & Kiley, P. J. (1995)."Characterization of FNR* mutant proteins indicates two distinct mechanisms for altering oxygen regulation of the Escherichia coli transcription factor FNR." Journal of bacteriology, 177(14), 3972.
3. Li, Songfeng, Delineation of Two Distinct Regulatory Domains in the 5' Region of the nar Operon of Escherichia coli. Journal of Bacteriology 164.1 (1985): 25-32.
4. Melville, Stephen. Isolation of an oxygen-sensitive FNR protein of Escherichia coli: Interaction at activator and repressor sites of FNR-controlled genes. Biochemistry 93. (1996): 1226-1231. Web. 6 Jun 2010.
5. Shaw, Duncan. "Nucleotide sequence of the fnr gene and primary structure of the Fnr protein of Escherichia coli." Nucleic Acids Research 10.19 (1982): 6119-6130. Web.