Part:BBa_K4271002
Antisense PhoU (AsPhoU)
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.
Usage in AsPhoU composite BBa_K4271003
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 (BBa_K4271002), 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
We cultured three different groups of E.coli DH5α (E.coli DH5α, E.coli DH5α with AsPhoU, E.coli DH5α with AsPhoU and arabinose) in both low and high phosphate concentrations. The groups cultured in low phosphate concentration act as the positive control of our preliminary experiment, while the groups cultured in high phosphate concentration function as the negative control.
For the experiment of malachite green coloration, we incubated E.coli DH5α (0.1 O.D.) with AsPhoU and E.coli DH5α with AsPhoU and arabinose under fixed high-phosphate environment (2 mM of K₂HPO₄, 0.06% glucose, and MOPS buffer). We retrieved our E.coli colonies respectively after 1 hour, 2 hours, and 3 hours of incubation in a high-phosphate environment. We then added molybdate and malachite into the tested groups and used a spectrometer to detect the absorbance of phosphate at 600 and 620 nm, since molybdate has a max absorbance rate at 600 nm while that of malachite is at 620 nm.
| Groups | Environmental Condition | Resulting coloration of E. coli colonies | ||
| E. coli DH5α | Low phosphate | Blue |  |
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| E. coli DH5α (without AsPhoU) | Low phosphate | Blue |  |
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| E. coli DH5α (with AsPhoU) + arabinose | Low phosphate | Blue |  |
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| E. coli DH5α | High phosphate | Transparent |  |
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| E. coli DH5α (with AsPhoU) | High phosphate | Transparent |  |
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| E. coli DH5α (with AsPhoU) + arabinose | High Phosphate | Blue |  |
Analysis of Results
From our preliminary result, we confirmed that the Pho regulon will only be active in a low phosphate environment as the positive control groups all turned blue, indicating PhoA enzyme activity and indicating PstSCAB activity. Our results also proved that arabinose has the ability to induce AsPhoU binding to the PhoU sequence, since the E. coli DH5α cultured with both AsPhoU and arabinose appears blue even in a high phosphate environment, showing PhoA and PstSCAB activity regardless of PhoU inhibition.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 208
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |