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| − | __NOTOC__
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| − | =Wild Type P<sub>kdpF</sub> with native RBS- potassium responsive promoter=
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| − | ===Biology of P<sub>kdpF</sub>===
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| − | [[File:HKUST-Rice_2015_K_MECHANISM2.jpg|thumb|500px|center|<b>Fig.1 </b>The Kdp K<sup>+</sup> uptake system in <i>E. coli</i> and the potassium biosensor design.]]
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| − | The potassium ion uptake in <i>E. coli</i> is regulated by several systems under different conditions. The potassium ion transporters, Trk and Kup are constitutively expressed (Epstein & Kim, 1971) while KdpFABC, another transporter is activated under low [K<sup>+</sup>] conditions (Laimins <i>et al.</i>, 1981).
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| − | The <i>kdpFABC</i> operon is controlled by the KdpDE two-component system (TCS) which consists of KdpD, a membrane-bound sensor kinase, and KdpE, a cytoplasmic response regulator (Polarek, 1992; Walderhaug, 1992). KdpD is stimulated by both intracellular and extracellular K<sup>+</sup> (Jung, 2000; Jung, 2001; Roe, 2000; Yan, 2011a; Laermann, 2013). KdpD phosphorylates KdpE upon low potassium concentration (Voelkner, 1993; Puppe, 1996; Jung, 1997a; Jung, 2000). Under an increase in [K<sup>+</sup>], KdpD phosphatase activity will be enhanced, causing a decrease in phospho-KdpE and <i>kdpFABC</i> expression. Phosphorylated KdpE turns on the expression of <i>kdpFABC</i> (Zhang, 2014a; Laermann, 2013).
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| − | ==Construction ==
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| − | [[File:HKUST-Rice 2015 kfig3.PNG|thumb|400px|center|'''''Fig 2.''' K<sup>+</sup> sensing construct with reporter.'']]
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| − | To make a potassium-sensing device, we obtained the promoter, P<sub>kdpF</sub>, and combined it with a GFP reporter, BBa_E0240, in BioBrick RFC10 standard so that the promoter activity in different potassium level can be detected and characterized.
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| − | ===Removal of <i>EcoR</i>I illegal site ===
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| − | To make P<sub>kdpF</sub> compatible with RFC10 standard and, as so, readily accessible to iGEM community, we designed variants of it with the <i>EcoR</i>I site removed. We mutated the thymine at -15 position to guanine, cytosine and adenine. The wild type promoter and the 3 variants are expected to be different in activity because of the difference in binding energy between the promoter and RNA polymerase (Brewster, 2012). Therefore, we characterized all of them to compare their strengths by relative fluorescence intensity, so as to obtain comprehensive knowledge in the activity and working range of the four promoters. For convenience, we denote them as A mutant, G mutant and C mutant respectively in the following context.
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| − | ==Characterization==
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| − | ===RPU measurement===
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| − | [[Image:HKUST-Rice15_(log_10)_RPU_of_kdpFp--15,T_G-_in_DH10B_-RPU-.png|thumb|700px|center|'''Fig6. Relative promoter unit (RPU) of P<sub>kdpF</sub>[-15,T>G] at different concentration of K<sup>+</sup>.''' ''Cells were pre-cultured in K115 medium overnight at 37°C. The cells were washed 3 times in 0.85% saline and then sub-cultured in medium with specific [K<sup>+</sup>]. Cells were fixed when OD<sub>600</sub> = 0.4. The measurement was carried out using fluorescence-activated cell sorting. Error bar are presented in SEM.'']]
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| − | In order to assemble a device that can be widely used by iGEM community, we characterized P<sub>kdpF</sub> by relative promoter unit (RPU) measurement. Additionally, relative fluorescence unit (RFU) measurement has also been done to compare the activities between wild type promoters and 3 variants.
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| − | At the lowest [K<sup>+</sup>], the strength of the G mutant promoter was found to be approximately 0.5 RPU. RPU of the promoter decreases as [K<sup>+</sup>] increases. At 0.025 mM [K<sup>+</sup>], RPU values was found to be 0.13. There was about 3.8 fold change in RPU from 0 mM to 0.4 mM [K<sup>+</sup>]. As expected, P<sub>kdpF</sub> is turned off at high [K<sup>+</sup>] due to inhibition of KdpD kinase activity by K<sup>+</sup>.
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| − | ===RFU measurement of 4 mutants===
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| − | [[File:HKUST-Rice_2015_4_promoter_RFU_+_GFP_syn_rate.png|thumb|900px|center|'''''Fig 3&4. Activity of P<sub>kdpF</sub> in <i>E. coli</i> DH10B in different K<sup>+</sup> concentrations''' Fluorescence/absorbance versus [K<sup>+</sup>] plot is shown on the left while the GFP synthesis rate versus [K<sup>+</sup>] plot is on the right. A, T(wild type), C, and G represent A mutant, wild type promoter, C mutant and G mutant respectively. Cells were pre-cultured, washed and sub-cultured as previously described in RPU measurement. Measurement took place when OD<sub>600</sub> = 0.4. 2 other measurements were taken every 15 mins afterwards for GFP synthesis rate. Error bar are presented in SEM.'' ]]
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| − | Both C and G mutants expressed higher fluorescence compared to the wild type and the A mutant. As the [K<sup>+</sup>] went up, the activity of P<sub>kdpF</sub> decreased. The expression levels of both the C and G mutant promoters were significantly higher than the wild type promoter, while the A mutant was always the lowest. At 0 mM [K<sup>+</sup>], both the C and G mutants expressed fluorescence which was about 1.7 times higher than the A mutant and wild type promoter. At 0.2 mM [K<sup>+</sup>], expression of both C and G mutants were 1.7 times higher than the wild type promoter, and 3 times higher compared to the A mutant. This might be caused by the difference in binding affinity between RNA polymerase and P<sub>kdpF</sub> variants (Brewster, 2012). The dynamic range of our promoters is between 0 to 0.1 mM of K<sup>+</sup>.
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| − | ===Promoter function measurement in different strain===
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| − | [[File:HKUST-Rice15_RFU_of_kdpFp_in_DH10B_and_TK2240.png|thumb|400px|center|'''Fig5.''' ''Comparison between the activities of P<sub>kdpF</sub>[-15, T>G] in DH10B and TK2240 strain. Cells were pre-cultured, washed and sub-cultured as previously described in RPU measurement. Measurement took place when OD<sub>600</sub> = 0.4. Error bars are represented as SEM.'' ]]
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| − | At [K<sup>+</sup>] lower than 0.0125 mM, the acitivity of G mutant in DH10B was significantly greater than that in TK2240 strain. Activity of P<sub>kdpF</sub> in DH10B strain decreased with increasing concentrations, while it remained stable in TK2240. Above 0.05 mM [K<sup>+</sup>], the activity of P<sub>kdpF</sub> in TK2240 strain exceeded that in the DH10B strain. Since TK2240 is defective in its Trk and Kup systems, it can only rely on the Kdp system for K<sup>+</sup> uptake, thereby explaining the higher promoter activity observed beyond 0.05 mM [K<sup>+</sup>].
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| − | <partinfo>BBa_K1682001 SequenceAndFeatures</partinfo>
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| − | <!-- Uncomment this to enable Functional Parameter display
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| − | ===Functional Parameters===
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| − | <partinfo>BBa_K1682001 parameters</partinfo>
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