Generator

Part:BBa_K256007:Design

Designed by: iGEM09_NTU-Singapore   Group: iGEM09_NTU-Singapore   (2009-10-16)

J23119:NorR


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 716
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 491


Design Notes

This construct is the actual system construct of our system. It is designed to generate NorR to prevent competitive binding of NorR.


Primer synthesis for NorR

  • Using NCBI’s reference sequence NC_000913.2 and E.coli (strain: K-12; substrain: MG1655) as search organism, the DNA sequence for NorR was obtained. The 1515 bp NorR resides at genome position 2828797 – 2830311, and the sequence is as follows:


ATGAGTTTTTCCGTTGATGTGCTGGCGAATATCGCCATCGAATTGCAGCGTGGGATTGGTCACCAGGATC GTTTTCAGCGCCTGATCACCACGCTACGTCAGGTGCTGGAGTGCGATGCGTCTGCGTTGCTACGTTACGA TTCGCGGCAGTTTATTCCGCTTGCCATCGACGGTCTGGCAAAGGATGTACTCGGTAGACGCTTTGCGCTG GAAGGGCATCCACGGCTGGAAGCGATTGCCCGCGCCGGGGATGTGGTGCGCTTTCCCGCAGACAGCGAAT TGCCCGATCCCTATGACGGTTTGATTCCTGGGCAGGAGAGTCTGAAGGTTCACGCCTGCGTTGGTCTGCC ATTGTTTGCCGGGCAAAACCTGATCGGCGCACTGACGCTCGACGGGATGCAGCCCGATCAGTTCGATGTT TTCAGCGACGAAGAGCTACGGCTGATTGCTGCGCTGGCGGCGGGAGCGTTAAGCAATGCGTTGCTGATTG AACAACTGGAAAGCCAGAATATGCTGCCAGGCGATGCCACGCCGTTTGAAGCGGTGAAACAGACGCAGAT GATTGGCTTGTCCCCTGGCATGACGCAACTGAAAAAAGAGATTGAGATTGTGGCGGCGTCCGATCTCAAC GTCCTGATCAGCGGTGAGACTGGAACCGGTAAGGAGCTGGTGGCGAAAGCGATTCATGAAGCCTCGCCAC GGGCGGTGAATCCGCTGGTCTATCTCAACTGTGCTGCACTGCCGGAAAGTGTGGCGGAAAGTGAGTTGTT CGGGCATGTGAAAGGAGCGTTTACTGGCGCTATCAGTAATCGCAGCGGGAAGTTTGAAATGGCGGATAAC GGCACGCTGTTTCTGGATGAGATCGGCGAGTTGTCGTTGGCATTGCAGGCCAAGCTGCTGAGGGTGTTGC AGTATGGCGATATTCAGCGCGTTGGCGATGACCGTTGTTTGCGGGTCGATGTGCGCGTGCTGGCGGCGAC TAACCGCGATTTACGCGAAGAGGTGCTGGCAGGGCGATTCCGCGCCGATTTGTTTCATCGCCTGAGCGTG TTTCCACTTTCGGTGCCGCCGCTGCGTGAGCGGGGCGATGATGTCATTCTGCTGGCGGGGTATTTCTGCG AGCAGTGTCGTTTGCGGCAGGGGCTCTCCCGCGTGGTATTAAGTGCCGGAGCGCGAAATTTACTGCAACA CTACAGTTTTCCGGGAAACGTGCGCGAACTGGAACATGCTATTCATCGGGCGGTAGTTCTGGCGAGAGCC ACCCGCAGCGGCGATGAAGTGATTCTTGAGGCGCAACATTTTGCTTTTCCTGAGGTGACGTTGCCGACGC CAGAAGTGGCGGCGGTGCCCGTTGTTAAGCAAAACCTGCGTGAAGCGACAGAAGCGTTCCAGCGTGAAAC TATTCGTCAGGCACTGGCACAAAATCATCACAACTGGGCTGCCTGCGCGCGGATGCTGGAAACCGACGTC GCCAACCTGCATCGGCTGGCGAAACGTCTGGGATTGAAGGATTAA


  • To ensure that the endogenous NorR sequence can be directly converted into a biobrick part without any modification, the sequence was checked with Webcutter 2.0 (http://rna.lundberg.gu.se/cutter2/) for any presence of EcoRI / NotI / XbaI / SpeI / PstI restriction sites. Since none of these restriction sites was found in the endogenouse NorR genome, direct PCR extraction can be performed. PCR primers were designed using NCBI’s PrimerBlast.


  • The primer sequences for NorR Extraction :
    • Forward Primer (27 bases): 5’-TTAATCCTTCAATCCCAGACGTTTCGC-3’
    • Reverse Primer (26 bases):5’- ATGAGTTTTTCCGTTGATGTGCTGGC-3’


  • Since NorR sequence is on the anti-sense strand of E.coli genome, the biobrick prefix has to be added to the reverse primer, rather than forward primer, and vice-versa.


  • The primer sequences for Biobrick conversion :
    • Addition of Preffix to Reverse primer – Forward Primer (50 bases) : 5’-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ATGAGTTTTTCCGTTGATGT-3’
    • Addition of Suffix to Forward primer – Reverse Primer (50 bases) : 5’-GTT TCT TCC TGC AGC GGC CGC TAC TAG TA TTAATCCTTCAATCCCAGACG-3’


The oligonucleotides were synthesized by one of our team’s sponsor, 1st Base Holdings, Singapore.

Source

NCBI’s reference sequence NC_000913.2.

The 1515 bp NorR resides at genome position 2828797 – 2830311

References

  • D’Autre´aux, B., N. P. Tucker, et al. (2005). "A non-haem iron centre in the transcription factor NorR senses nitric oxide." Nature 437(29): 769 -772.
  • Hutchings, M. I., N. Mandhana, et al. (2002). "The NorR protein of Escherichia coli activates expression of the Flavorbredoxin gene NorV in response to reactive nitrogen species." Journal of bacteriology 184(16): 4640 - 4643.
  • Tucker, N. P., B. D. Autreaux, et al. (2006). "Mechanism of transcriptional regulation by Escherichia Coli nitric oxide sensor NorR." Biochemical Society Transactions 34(1): 191 - 194.
  • Ferenc I. Tarr MD, P., Mária Sasvári MDb, Márton Tarr MD and Rozália Rácz MD (2005). "Evidence of Nitric Oxide Produced by the Internal Mammary Artery Graft in Venous Drainage of the Recipient Coronary Artery." The Annals of Thoracic Surgery 80(5): 1728-1731.