Part:BBa_K4271003
araBAD promoter + RBS + AsPhoU + T1 T2 terminator
Design
The araBAD promoter is a sequence present in the pBAD vector that is activated by 0.2% of arabinose. This is used as positive control in our experiment to ensure downstream AsPhoU expression, whose correlation will be later described.
PstSCAB is a high-affinity phosphate transporter protein in E. coli that allows phosphate from entering the cell. In low inorganic phosphate conditions, PhoU, a metal binding protein that detects inorganic phosphate levels, will dissociate from the PstSCAB transporter, thus allowing phosphate to enter the cell. On the contrary, during in increase of phosphate in the environment, PhoU will bind to PstSCAB and inhibit Pi transportation. In order to combat eutrophication, a phenomenon caused by increase of nitrogen and phosphate in bodies of water, we designed the AsPhoU gene, which encodes antisense PhoU RNA against phoU expression, to increase the amount of Pi that bacteria could transport; thus lowering the concentration of Pi in aquatic environments. The engineered antisense PhoU DNA in our bacteria would be transcribed into antisense PhoU RNA(AsPhoU), which would then bind to the mRNA of PhoU, hindering ribosome binding to decrease phoU translation. The inhibition of PhoU protein would allow the PstSCAB transporter to be open for Pi transportation at all times, even under the high concentration of phosphate in eutrophic water bodies.
The Ti and T2 terminator from the region of the rrrnB gene in E. coli are strong terminator that prevent leaky expressions.
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Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1144
Illegal XhoI site found at 1442 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
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