Part:BBa_K1422505:Design
Lactose-inducible killswitch, produces SAMS and NAS1
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2436
Illegal PstI site found at 1120
Illegal PstI site found at 1606 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2436
Illegal NheI site found at 1566
Illegal PstI site found at 1120
Illegal PstI site found at 1606 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2436
Illegal BglII site found at 1056
Illegal XhoI site found at 1940 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2436
Illegal PstI site found at 1120
Illegal PstI site found at 1606 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2436
Illegal PstI site found at 1120
Illegal PstI site found at 1606
Illegal NgoMIV site found at 457
Illegal NgoMIV site found at 944
Illegal NgoMIV site found at 1739
Illegal AgeI site found at 3667
Illegal AgeI site found at 3779 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1943
Design Notes
The kill switch is located at the beginning of a plasmid to avoid being accidentally translated by an mRNA due to a previous section not being properly terminated. Using a conditional promoter so that the modified Bacillus subtilis would only produce phytosiderophores in the presence of plants roots was considered, but it was decided that conditional promoters would not be reliable enough. A constitutive promoter of medium strength was chosen for the corn genes because a weak promoter might not have produced enough phytosiderophores to make a difference, and a strong promoter might have overtaxed the cell in the production of phytosiderophores as to be counterproductive. The RBS SpoVG was chosen for its relative strength in Bacillus subtilis. The relatively strong terminator B1006 was selected for its relative effectiveness and low number of base pairs. The reporter is placed after the corn genes because gene expression decreases linearly as the distance from the promoter increases, and expressing the corn genes is much more important than expressing the reporter. The corn genes are codon optimized for Bacillus subtilis (details are on their respective Registry pages). NAS can be produced from the genes nas1, nas2, nas3, nas4, nas5-1, nas5-2, and nas6. nas1 was selected because in a previous experiment it was able to, alone, produce NAS.
Source
Many of the genes (Hyb5, SAMS, and NAS1) come fromNCBI. (2 December, 2008). Lactobacillus phage YB5 holin gene, complete cds. http://www.ncbi.nlm.nih.gov/nuccore/EF623894?report=GenBank NCBI. NCBI. (2014). S-adenosylmethionine synthetase 1 [Zea mays]. http://www.ncbi.nlm.nih.gov/protein/NP_001148708.1 NCBI. (5 February, 2003). Zea mays ZmNAS1 mRNA, complete cds. http://www.ncbi.nlm.nih.gov/nuccore/19911063