Difference between revisions of "Part:BBa K3560005"
 
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We successfully verified three important phosphate dissolution systems in the project. Phosphate dissolving system shows considerable ability to dissolve phosphate. As time goes by, the pH value of the solution gradually decreases, and the dissolving range of phosphate becomes larger. In addition, the Phosphate dissolving Plus system also shows a more efficient ability to dissolve phosphate. As for the GDH-Pro system, we strengthen the expression of GDH, increase the effective concentration of enzymes, and promote the dissolution of phosphate. The functions of the above-mentioned systems are all realized in Deinococcus radiodurans(DR), which meets the needs of soil modification on Mars.
 
We successfully verified three important phosphate dissolution systems in the project. Phosphate dissolving system shows considerable ability to dissolve phosphate. As time goes by, the pH value of the solution gradually decreases, and the dissolving range of phosphate becomes larger. In addition, the Phosphate dissolving Plus system also shows a more efficient ability to dissolve phosphate. As for the GDH-Pro system, we strengthen the expression of GDH, increase the effective concentration of enzymes, and promote the dissolution of phosphate. The functions of the above-mentioned systems are all realized in Deinococcus radiodurans(DR), which meets the needs of soil modification on Mars.
 
<h2> Experiment and Results </h2>
 
<h2> Experiment and Results </h2>
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Phosphate dissolving Plus system in Deinococcus radiodurans:
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Phosphate dissolving Plus system in Deinococcus radiodurans
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We design an enhanced Phosphate dissolving system(Phosphate dissolving Plus system) has two plasmids：gcd-pRADK, gabY-pRADK. gabY gene circuit has four parts: PGroES(BBa_K3560002), RBS(BBa_K3560003), gabY(BBa_K3560001), TT(BBa_B0015) (figure 1). In order to realize the function of gene circuit, we construct the following plasmid gabY-pRADK (figure 2). And we verified the length of the recombinant plasmid to ensure the success of the recombinant plasmid through enzyme digestion (figure 3). Theoretically, after transforming gabY-pRADK to DR containing gcd-pRADK, the effective concentration of the whole enzyme formed by GDH and PQQ coenzymes will increase, and the catalytic efficiency will be higher.  
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We design an enhanced Phosphate dissolving system (Phosphate dissolving Plus system) which has two plasmids：gcd-pRADK, gabY-pRADK. gabY gene circuit has four parts: PGroES(BBa_K3560002), RBS(BBa_K3560003), gabY(BBa_K3560001), TT(BBa_B0015) (Figure 1). In order to realize the function of gene circuit, we construct the following plasmid gabY-pRADK (Figure 2). And we verified the length of the recombinant plasmid to ensure the success of the recombinant plasmid through enzyme digestion (Figure 3). Theoretically, after transforming gabY-pRADK to DR containing gcd-pRADK, the effective concentration of the holoenzyme formed by GDH and PQQ coenzymes will increase, and the catalytic efficiency will be higher.  
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<li style="display: inline-block;"> [[File: T--XHD-Wuhan-China--parts-figure1gabY.png |thumb|none|500px]]
 
 
Figure 1. Constitution of PGroES-RBS-gabY gene circuits.
 
Figure 1. Constitution of PGroES-RBS-gabY gene circuits.
  
  
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<li style="display: inline-block;"> [[File: T--XHD-Wuhan-China--parts-figure2gabY.png |thumb|none|500px]]
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Figure 2. Design of Phosphate dissolving Plus plasmid (gabY-pRADK).
 
Figure 2. Design of Phosphate dissolving Plus plasmid (gabY-pRADK).
  
  
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<li style="display: inline-block;"> [[File: T--XHD-Wuhan-China--parts-figure3gabY.png |thumb|none|500px]]
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  <img src="https://static.igem.org/mediawiki/parts/a/a3/T--XHD-Wuhan-China--parts-figure3gabY.jpeg" style="width:60%">
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Figure 3. ElectropHoresis of plasmid gabY-pRADK with enzyme digestions.
 
Figure 3. ElectropHoresis of plasmid gabY-pRADK with enzyme digestions.
  
  
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We cultured DR containing gcd-pRADK, gabY-pRADK; DR containing gcd-pRADK; and wild-type DR in TGY liquid medium, and measured pH every 1 hour. The results are shown in the figure 4. In addition, DR containing gcd-pRADK, gabY-pRADK; DR containing gcd-pRADK; and wild-type DR were cultured in PKO solid medium, and the results of the phosphate ring were observed, as shown in the figure 5.
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We cultured DR containing gcd-pRADK, gabY-pRADK; DR containing gcd-pRADK; and wild-type DR in TGY liquid medium, and measured pH every 1 hour. The results are shown in the Figure 4.
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  <img src="https://static.igem.org/mediawiki/parts/8/86/T--XHD-Wuhan-China--parts-figure4gabY.png" style="width:60%">
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Figure 4. Changes in pH value of TGY liquid medium culturing DR R1, DR containing gcd-pRADK and DR containing pRADK2 respectively.
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<h2>Conclusion</h2>
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The above results show that we have successfully constructed the Phosphate dissolving Plus system in DR, and compared with phosphate dissolution systems, the Phosphate dissolving Plus system has a stronger pH decrease speed and range, and has a stronger ability to dissolve phosphate. Compared to phosphate dissolution systems, Phosphate dissolving Plus system is more efficient in decreasing pH and dissolving phosphate. It can dissolve more phosphate in a shorter time and improve the efficiency of transforming Martian soil. In addition, this system is more suitable for soils with more insoluble phosphates.
  
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<li style="display: inline-block;"> [[File: T--XHD-Wuhan-China--parts-figure4gabY.png |thumb|none|500px]]
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<h2> References</h2>
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Figure 4. Changes in pH value of TGY liquid medium culturing D.r R1, D.r containing gcd-pRADK and D.r containing pRADK2 respectively.
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Ahemad, Munees. Phosphate-solubilizing bacteria-assisted phytoremediation of metalliferous soils: a review[J]. Biotech, 2015, 5(2):111-121.
  
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Adcock C T , Hausrath E M , Forster P M . Readily available phosphate from minerals in early aqueous environments on Mars[J]. Nature Geoence, 2013, 6(10):824-827.
  
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<li style="display: inline-block;"> [[File: T--XHD-Wuhan-China--parts-figure5gabY.jpeg |thumb|none|500px]]
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Clifton K P , Jones E M , Paudel S , et al. The genetic insulator RiboJ increases expression of insulated genes[J]. Journal of Biological Engineering, 2018, 12(1).
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Figure 5. Phosphate ring in PKO solid medium culturing D.r R1, D.r containing gcd-pRADK and D.r containing pRADK2 respectively. Left: D.r R1; Middle: D.r containing gcd-pRADK; Right: D.r containing pRADK2.
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Latest revision as of 14:04, 27 October 2020


PGroES-DrRBS-gabY

gabY promotes the combination of PQQ coenzyme and GDH, increases the effective concentration of the holoenzymes, and enhances its catalytic efficiency. The gabY fragment was recombined with pRADK to form gabY-pRADK, Transform gabY-pRADK into DR containing gcd-pRADK plasmids, so that DR containing gabY-pRADK and gcd-pRADK plasmids can decrease the pH to a greater extent and promote the dissolution of phosphate.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 393
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 190
    Illegal NgoMIV site found at 307
    Illegal NgoMIV site found at 399
    Illegal NgoMIV site found at 565
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

We successfully verified three important phosphate dissolution systems in the project. Phosphate dissolving system shows considerable ability to dissolve phosphate. As time goes by, the pH value of the solution gradually decreases, and the dissolving range of phosphate becomes larger. In addition, the Phosphate dissolving Plus system also shows a more efficient ability to dissolve phosphate. As for the GDH-Pro system, we strengthen the expression of GDH, increase the effective concentration of enzymes, and promote the dissolution of phosphate. The functions of the above-mentioned systems are all realized in Deinococcus radiodurans(DR), which meets the needs of soil modification on Mars.

Experiment and Results

Phosphate dissolving Plus system in Deinococcus radiodurans

We design an enhanced Phosphate dissolving system (Phosphate dissolving Plus system) which has two plasmids：gcd-pRADK, gabY-pRADK. gabY gene circuit has four parts: PGroES(BBa_K3560002), RBS(BBa_K3560003), gabY(BBa_K3560001), TT(BBa_B0015) (Figure 1). In order to realize the function of gene circuit, we construct the following plasmid gabY-pRADK (Figure 2). And we verified the length of the recombinant plasmid to ensure the success of the recombinant plasmid through enzyme digestion (Figure 3). Theoretically, after transforming gabY-pRADK to DR containing gcd-pRADK, the effective concentration of the holoenzyme formed by GDH and PQQ coenzymes will increase, and the catalytic efficiency will be higher.

Figure 1. Constitution of PGroES-RBS-gabY gene circuits.


Figure 2. Design of Phosphate dissolving Plus plasmid (gabY-pRADK).


Figure 3. ElectropHoresis of plasmid gabY-pRADK with enzyme digestions.


We cultured DR containing gcd-pRADK, gabY-pRADK; DR containing gcd-pRADK; and wild-type DR in TGY liquid medium, and measured pH every 1 hour. The results are shown in the Figure 4.

Figure 4. Changes in pH value of TGY liquid medium culturing DR R1, DR containing gcd-pRADK and DR containing pRADK2 respectively.


Conclusion

The above results show that we have successfully constructed the Phosphate dissolving Plus system in DR, and compared with phosphate dissolution systems, the Phosphate dissolving Plus system has a stronger pH decrease speed and range, and has a stronger ability to dissolve phosphate. Compared to phosphate dissolution systems, Phosphate dissolving Plus system is more efficient in decreasing pH and dissolving phosphate. It can dissolve more phosphate in a shorter time and improve the efficiency of transforming Martian soil. In addition, this system is more suitable for soils with more insoluble phosphates.

References

Ahemad, Munees. Phosphate-solubilizing bacteria-assisted phytoremediation of metalliferous soils: a review[J]. Biotech, 2015, 5(2):111-121.

Adcock C T , Hausrath E M , Forster P M . Readily available phosphate from minerals in early aqueous environments on Mars[J]. Nature Geoence, 2013, 6(10):824-827.

Clifton K P , Jones E M , Paudel S , et al. The genetic insulator RiboJ increases expression of insulated genes[J]. Journal of Biological Engineering, 2018, 12(1).