Difference between revisions of "Part:BBa K1682000"

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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).
 
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).
 
<br><br>
 
<br><br>
===Construct for characterization ===
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==Construct for characterization ==
  
 
[[Image:HKUST-Rice 2015 kfig3.PNG|300px|center]]
 
[[Image:HKUST-Rice 2015 kfig3.PNG|300px|center]]
 +
 
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.
 
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 <i>EcoR</i>I illegal site ===
 
===Removal of <i>EcoR</i>I 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.
 
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.
 +
 +
  
 
===RFU measurement of 4 mutants===
 
===RFU measurement of 4 mutants===
 
[[Image:HKUST-Rice_2015_4_promoter_RFU_+_GFP_syn_rate.png|900px|center]]
 
[[Image:HKUST-Rice_2015_4_promoter_RFU_+_GFP_syn_rate.png|900px|center]]
 
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+.
 
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+.
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 +
<br><br>
  
  
 
===Promoter function measurement in different strain===
 
===Promoter function measurement in different strain===
[[Image:HKUST-Rice15_RFU_of_kdpFp_in_DH10B_and_TK2240.png|600px|right]]
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[[Image:HKUST-Rice15_RFU_of_kdpFp_in_DH10B_and_TK2240.png|400px|right]]
 
At [K+] lower than 0.0125 mM, the acitivity of G mutant in DH10B was significantly greater than that in TK2240 strain. Activity of PkdpF in DH10B strain decreased with increasing concentrations, while it remained stable in TK2240. Above 0.05 mM [K+], the activity of PkdpF 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+ uptake, thereby explaining the higher promoter activity observed beyond 0.05 mM [K+].
 
At [K+] lower than 0.0125 mM, the acitivity of G mutant in DH10B was significantly greater than that in TK2240 strain. Activity of PkdpF in DH10B strain decreased with increasing concentrations, while it remained stable in TK2240. Above 0.05 mM [K+], the activity of PkdpF 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+ uptake, thereby explaining the higher promoter activity observed beyond 0.05 mM [K+].
 +
 +
<br><br>
  
 
===RPU measurement===
 
===RPU measurement===
[[Image:HKUST-Rice15_(log_10)_RPU_of_kdpFp--15,T_G-_in_DH10B_-RPU-.png|900px|center]]
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[[Image:HKUST-Rice15_(log_10)_RPU_of_kdpFp--15,T_G-_in_DH10B_-RPU-.png|700px|center]]
 
In order to assemble a device that can be widely used by iGEM community, we characterized PkdpF 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.
 
In order to assemble a device that can be widely used by iGEM community, we characterized PkdpF 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.
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1682000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1682000 SequenceAndFeatures</partinfo>
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Revision as of 18:44, 13 September 2015

Wild Type PkdpF - potassium responsive promoter

Biology of PkdpF

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

The 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).


Construct for characterization

HKUST-Rice 2015 kfig3.PNG

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.


RFU measurement of 4 mutants

HKUST-Rice 2015 4 promoter RFU + GFP syn rate.png

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+.




Promoter function measurement in different strain

HKUST-Rice15 RFU of kdpFp in DH10B and TK2240.png

At [K+] lower than 0.0125 mM, the acitivity of G mutant in DH10B was significantly greater than that in TK2240 strain. Activity of PkdpF in DH10B strain decreased with increasing concentrations, while it remained stable in TK2240. Above 0.05 mM [K+], the activity of PkdpF 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+ uptake, thereby explaining the higher promoter activity observed beyond 0.05 mM [K+].



RPU measurement

HKUST-Rice15 (log 10) RPU of kdpFp--15,T G- in DH10B -RPU-.png

In order to assemble a device that can be widely used by iGEM community, we characterized PkdpF 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.




Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 61
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 61
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 61
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 61
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 61
  • 1000
    COMPATIBLE WITH RFC[1000]