Difference between revisions of "Part:BBa K4271003"

Revision as of 05:40, 8 October 2022

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.

Build

In order to determine the amount of phosphate entering the bacteria, we utilized certain components of the PhoU regulon to measure the effectiveness of phosphate transportation. To evaluate the activity of the PstSCAB transporter, we conducted a preliminary experiment that measures the concentration of PhoA via its coloration in low and high phosphate environments. Since the activity of PstSCAB and PhoA are positively correlated, an increase in PhoA concentration will indicate the activity of the PstSCAB transporter. In this preliminary experiment, we added solutions of 5-Bromo-4-chloro-3-indolyl phosphate (XP) because PhoA will severe it into a phosphate ion and a 5,5′-dibromo-4,4′-dichloro-indigo, which makes the solution blue. Arabinose also plays an important role in our preliminary experiment, since it acts as an inducer that promotes AsPhoU to bind on the PhoU sequence.

Another experiment we conducted to measure the effectiveness of phosphate transportation into the cell is to measure the amount of extracellular phosphate in the bacteria via malachite green coloration. A complex of phosphomolybdic acid is formed when molybdate (MoO₄⁻²) interacts with phosphate (PO₄⁻³), which would later interact with malachite and form a green chromogenic complex.

Test

	Groups	Environmental Condition	Resulting coloration of E. coli colonies
E. coli DH5α	Low phosphate	Blue
E. coli DH5α (without AsPhoU)	Low phosphate	Blue
E. coli DH5α (with AsPhoU) + arabinose	Low phosphate	Blue
E. coli DH5α	High phosphate	Transparent
E. coli DH5α (with AsPhoU)	High phosphate	Transparent
E. coli DH5α (with AsPhoU) + arabinose	High Phosphate	Blue

Sequence and Features