Part:BBa_K1962014
A pH Sensing Device Based on The Asr Promoter
The "acid shock RNA" asr promoter is a pH sensitive promoter native to E. coli that has been documented by other iGEM teams. Here, we present a composite part to allow further characterisation of promoter activity. The composite part consists of the asr promoter(BBa_K1231000) and a combined biobrick containing RBS / GFP / terminators (BBa_E0840).
s===Usage and Biologys===
Enterobacteria can respond to low pH by de novo synthesis of specific proteins and altered levels of gene expression. The response to environmental stresses, such as pH, is often a complex mechanism and also depends on other environmental factors such as nutrition, the presence or absence of oxygen or starvation. The E. coli asr (acid shock RNA) gene encodes small RNA, of about 450 nucleotides in length. This gene is inducible by low external pH and contributes to the organism’s survival2. It has been suggested that asr genes may be regulated by the two component system PhoBR2.
In this two-component system the protons from the environment (H+) activate the sensory part (PhoR- in the periplasm) of the two-component system, which then transduces the signal to the activator protein- PhoB, which can bind promoter DNA of asr. The promoter region of asr was analysed and showed to contain a sequence similar to the pho-box; this is a consensus sequence able to bind to the PhoB protein2. These interactions of H+ from the external environment with this two-component system are thought to lead to asr transcription. Interestingly the PhoBR system also controls the pho regulon which is induced by phosphate starvation, the link between these two factors is not fully understood however, it has been suggested that the level of Asr expressed in minimal media is higher than in enriched media.
In 2013 the Northwestern iGEM team submitted two pH sensitive promoter Pasr (BBa_K1231000) and PgadA (BBa_K1231001). In order to further characterise both of these promoters we cloned gfp (BBa_E0840) downstream of both promoters. We then used this construct to measure and compare the GFP expression levels in response to different pH conditions.
We wanted to test both our pH sensitive promoters, Pasr and PgadA by monitoring for the production of GFP by western blotting. The western blots showed levels of GFP protein production by the cells at a range of pH values. In Fig 1A the expression of GFP at pH 5 is much higher than the other pH readings. This would indicate that at this pH the largest amount of GFP was being produced under the induction of the gadA promoter. The promoter is slightly leaky as there is also expression of protein within cells at other pH values. In Fig 1B the GFP protein expression under the regulation of Pasr can be seen, the range of activity for this promoter is much wider indicating that this promoter is leakier than PgadA. The level of expression of GFP under the regulation of Pasr appears to be uniform with only a slight increase in GFP at pH5.
Fig 1. GFP production under the control of pH sensitive promoters PgadA and Pasr. Promoters with gfp cloned downstream of them were transformed into MG1655 E. coli cells, 5 ml of these cells were incubated for 16h at 37 ° C. After 16h, 200 uL of the overnight cultures were introduced into 1.8 ml of MOPS pH adjusted LB for the respective pH values indicated above. After 20 min at ambient conditions a 1ml aliquot was pelleted. The pellets were re-suspended in 100 ul Laemmli buffer and 15uL samples were then separated by SDS PAGE (12% acrylamide) and transferred to PVDF membrane followed by probing with anti-GFP antibody. Fig 2A - Shows the GFP production under the control of the PgadA and Fig 2B shows the GFP production under the control of the Pasr promoter both in varying pH conditions.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 813
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