Difference between revisions of "Part:BBa K1682003"

 
 
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<partinfo>BBa_k1682003 short</partinfo>
  
__NOTOC__
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C-mutant <i>P<sub>kdpF</sub></i> - potassium responsive promoter
<partinfo>BBa_K1682003 short</partinfo>
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kdpFp[-15, T>C]
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===Biology of <i>P<sub>kdpF</sub></i>===
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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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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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<br><br>
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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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<br><br>
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We adopt the promoter <i>P<sub>kdpF</sub></i> from <i>kdpFABC</i> operon with -28 position of transcription start site relative to start the first gene, <i>kdpF</i>. The -10 and -35 box elements have been mapped which are TACCCT and TTGCGA respectively (Sugiura et al., 1992). The transcription factor, phosphorylated KdpE, binds to the <i>P<sub>kdpF</sub></i> at binding site located from -71 to -55 site with reference to the transcription start site (Sugiura et al., 1992; Narayanan et al., 2012).
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<br><br>
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==Construction==
  
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[[File:HKUST-Rice 2015 kfig3.PNG|thumb|400px|center|<b>Fig 2.</b> K+ sensing construct with reporter.]]
===Usage and Biology===
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To make a potassium-sensing device, we obtained the promoter, <i>P<sub>kdpF</sub></i>, 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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<br><br>
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===Removal of <i>EcoR</i>I illegal site ===
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To make <i>P<sub>kdpF</sub></i> 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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<br><br>
  
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==Characterization==
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1682003 SequenceAndFeatures</partinfo>
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===RFU measurement of 3 mutants===
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[[File:HKUST-Rice_2015_4_promoter_RFU_+_GFP_syn_rate.png|thumb|700px|center|<b>Fig 4.  Activity of <i>P<sub>kdpF</sub></i> in <i>E. coli</i> DH10B in different K<sup>+</sup> concentrations</b> A) Positions of base substitutions to standardize <i>P<sub>kdpF</sub></i> into RFC10 format. B) Single time point transfer curve for <i>P<sub>kdpF</sub></i> variants along a gradient of [K+]. C) Relative GFP synthesis rate calculated from 3 measurement time points. 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 present SEM from 3 biological replicates.]]
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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 <i>P<sub>kdpF</sub></i> 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 <i>P<sub>kdpF</sub></i> variants (Brewster, 2012). The dynamic range of our promoters is between 0 to 0.1 mM of K<sup>+</sup>.
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<br><br>
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<partinfo>BBa_K1682003 SequenceAndFeatures</partinfo>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 04:30, 19 September 2015

PkdpF[-15, T>C]

C-mutant PkdpF - potassium responsive promoter

Biology of PkdpF

Fig 1. The Kdp K+ uptake system in E. coli and the potassium biosensor design.

Potassium ion uptake in E. coli 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+] conditions (Laimins et al., 1981).

The kdpFABC 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+ (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+], KdpD phosphatase activity will be enhanced, causing a decrease in phospho-KdpE and kdpFABC expression. Phosphorylated KdpE turns on the expression of kdpFABC (Zhang, 2014a; Laermann, 2013).

We adopt the promoter PkdpF from kdpFABC operon with -28 position of transcription start site relative to start the first gene, kdpF. The -10 and -35 box elements have been mapped which are TACCCT and TTGCGA respectively (Sugiura et al., 1992). The transcription factor, phosphorylated KdpE, binds to the PkdpF at binding site located from -71 to -55 site with reference to the transcription start site (Sugiura et al., 1992; Narayanan et al., 2012).

Construction

Fig 2. K+ sensing construct with reporter.

To make a potassium-sensing device, we obtained the promoter, PkdpF, 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.

Removal of EcoRI illegal site

To make PkdpF compatible with RFC10 standard and, as so, readily accessible to iGEM community, we designed variants of it with the EcoRI 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.

Characterization

RFU measurement of 3 mutants

Fig 4. Activity of PkdpF in E. coli DH10B in different K+ concentrations A) Positions of base substitutions to standardize PkdpF into RFC10 format. B) Single time point transfer curve for PkdpF variants along a gradient of [K+]. C) Relative GFP synthesis rate calculated from 3 measurement time points. Cells were pre-cultured, washed and sub-cultured as previously described in RPU measurement. Measurement took place when OD600 = 0.4. 2 other measurements were taken every 15 mins afterwards for GFP synthesis rate. Error bar present SEM from 3 biological replicates.

Both C and G mutants expressed higher fluorescence compared to the wild type and the A mutant. As the [K+] went up, the activity of PkdpF 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+], 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+], 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 PkdpF variants (Brewster, 2012). The dynamic range of our promoters is between 0 to 0.1 mM of K+.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]