Regulatory

Part:BBa_K1484339:Design

Designed by: Angelina Munabi   Group: iGEM14_Cambridge-JIC   (2014-10-10)


P_NiI23, marchantia promoter


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 601
    Illegal EcoRI site found at 1816
    Illegal XbaI site found at 791
    Illegal XbaI site found at 1906
    Illegal PstI site found at 1525
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 601
    Illegal EcoRI site found at 1816
    Illegal PstI site found at 1525
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 601
    Illegal EcoRI site found at 1816
    Illegal BamHI site found at 44
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 601
    Illegal EcoRI site found at 1816
    Illegal XbaI site found at 791
    Illegal XbaI site found at 1906
    Illegal PstI site found at 1525
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 601
    Illegal EcoRI site found at 1816
    Illegal XbaI site found at 791
    Illegal XbaI site found at 1906
    Illegal PstI site found at 1525
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 105
    Illegal SapI.rc site found at 1572


Design Notes

The removal of illegal restriction sites was considered but not completed due to direct interaction of the DNA with regulatory proteins. Also, it was ensured that the region selected for this part was directly upstream of the first ATG of the gene used to identify the promoter sequence. This was so that the 5' UTR, that is vital in plants, was maintained.


Source

This part was found by screening the genome of the Marchantia polymorpha Cam strain maintained by the Haseloff lab for homologues to a Nitrate Transporter protein in A. Thaliana known as AtNRT2.1 or ACH1. This is a putative high-affinity nitrate transporter, potentially expressed slightly later in development. Transcription is thought to be induced by very low levels of nitrate (50 μM KNO3). Matches to the protein CDS sequence were found using Geneious to perform a tblastn search on the genome scaffolds. Predicted genes that contained hits graded above 30% and with at least 40% congruence to mRNA transcript sequences were shortlisted. The best gene candidates (judged according to number and distribution of hits along its length, and supporting mRNA sequence) formed the basis for our predicted promoters. This part was identified as a 2kb region upstream of the first ATG of such a gene.


References

Forde BG. 2000. Nitrate transporters in plants: structure, function and regulation. (BBA) – Biomembranes 1465(1-2):219-235. Filleur S, Daniel-Vedele F. 1998. Expression analysis of a high-affinity nitrate transporter isolated from Arabidopsis thaliana by differential display. Planta 207: 461-469. Thalemine, ATG id AT1G08090