Difference between revisions of "Part:BBa K2717007"
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===<h1>2020 XHD-Wuhan-China's Improvement</h1>===
 
===<h1>2020 XHD-Wuhan-China's Improvement</h1>===
 
<h2> Introduction </h2>
 
<h2> Introduction </h2>
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BBa_K2717007 contains RBS and gcd genes. We plan to improve this part. We inserted a RiboJ sequence in the upstream of gcd and downstream of the PGroES promoter. In addition, we described the functions of this part in Deinococcus radiodurans (DR), including the growth curve and the ability to lower pH, and successfully constructed GDH-Pro system.
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BBa_K2717007 contains RBS and gcd genes. We plan to improve this part, and constructed improved part <partinfo>K3560006 </partinfo>. We inserted a RiboJ sequence in the upstream of gcd and downstream of the PGroES promoter. In addition, we described the functions of this part in Deinococcus radiodurans (DR), including the growth curve and the ability to decrease pH and dissolve phosphate.
 
<h2> Experiment and Results </h2>
 
<h2> Experiment and Results </h2>
 
GDH-Pro system
 
GDH-Pro system
  
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We designed a gcd gene expression enhancement system, inserting the RiboJ sequence downstream of the PGroES promoter, and exposing RBS during the translation process to enhance the expression of the gcd gene. The circuit has four parts: PGroES(BBa_K3560002), RiboJ(BBa_K2066535), RBS(BBa_K3560003), gcd(BBa_K2717000), TT(BBa_B0015) (figure 1). We obtained gcd-pRADK-RiboJ plasmid through homologous recombination on the basis of gcd-pRADK (figure 2), and then transformed it into DR. And we verified the length of the recombinant plasmid to ensure the success of the recombinant plasmid through enzyme digestion (figure 3). Theoretically, the DR containing the gcd-pRADK-RiboJ plasmid will express more GDH, will have a higher catalytic efficiency, a greater decrease in pH, and a greater range of phosphate ring.  
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We designed a gcd gene expression enhancement system, inserting the RiboJ sequence downstream of the PGroES promoter, and exposing RBS during the translation process to enhance the expression of the gcd gene. The circuit has four parts: PGroES(BBa_K3560002), RiboJ(BBa_K2066535), RBS(BBa_K3560003), gcd(BBa_K2717000), TT(BBa_B0015) (Figure 1). We obtained gcd-pRADK-RiboJ plasmid through homologous recombination on the basis of gcd-pRADK (Figure 2), and then transformed it into DR. And we verified the length of the recombinant plasmid to ensure the success of the recombinant plasmid through enzyme digestion (Figure 3). Theoretically, the DR containing the gcd-pRADK-RiboJ plasmid will express more GDH, will have a higher catalytic efficiency, a greater decrease in pH, and a greater range of phosphate ring.  
  
 
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We cultured DR R1, DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ respectively in TGY liquid medium, and then determined the growth curve. As shown in Figure 4, DR with high GDH expression has a faster growth rate. Then, we cultured DR containing gcd-pRADK-RiboJ and DR containing gcd-pRADK in TGY liquid medium respectively, and measured the pH every 1 hour. The results are shown in the figure 5. In addition, the DR containing gcd-pRADK-RiboJ and the DR containing gcd-pRADK were cultured in PKO solid medium, and the results of the phosphate ring were observed, as shown in the figure 6.
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We cultured DR R1, DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ respectively in TGY liquid medium, and then determined the growth curve. As shown in Figure 4, DR with high GDH expression has a faster growth rate. Then, we cultured DR containing gcd-pRADK-RiboJ and DR containing gcd-pRADK in TGY liquid medium respectively, and measured the pH every 1 hour. The results are shown in the Figure 5. In addition, the DR containing gcd-pRADK-RiboJ and the DR containing gcd-pRADK were cultured in PKO solid medium, and the results of the phosphate ring were observed, as shown in the Figure 6.
  
 
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Revision as of 14:05, 27 October 2020


RBS2000-gdh-v5 tag-his tag

This part is a composite one, in which Promoter lacI and RBS 2000 control the glucose dehydrogenase and V5 tag and His tag. Rbs2000 can strengthen the expression of glucose dehydrogenase. Tag can be used to purify the proteins and to detect the expression of proteins. We constructed this vector to verify the growth-promoting effect of glucose dehydrogenase and its ability to retain plasmids.

function:

We designed this part can make gdh overexpressed due to RBS2000, and purify the glucose dehydrogenase protein with the help of the V5 tag and His tag and detect its expression. After confirming the expression of gdh, we used the same strain, and under the induction of IPTG, quantitatively detected the growth of the cells and the retention of the plasmid to verify the growth-promoting effect and plasmid retention of glucose dehydrogenase.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1569
    Illegal AgeI site found at 2461
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2443



2020 XHD-Wuhan-China's Improvement

Introduction

BBa_K2717007 contains RBS and gcd genes. We plan to improve this part, and constructed improved part BBa_K3560006. We inserted a RiboJ sequence in the upstream of gcd and downstream of the PGroES promoter. In addition, we described the functions of this part in Deinococcus radiodurans (DR), including the growth curve and the ability to decrease pH and dissolve phosphate.

Experiment and Results

GDH-Pro system

We designed a gcd gene expression enhancement system, inserting the RiboJ sequence downstream of the PGroES promoter, and exposing RBS during the translation process to enhance the expression of the gcd gene. The circuit has four parts: PGroES(BBa_K3560002), RiboJ(BBa_K2066535), RBS(BBa_K3560003), gcd(BBa_K2717000), TT(BBa_B0015) (Figure 1). We obtained gcd-pRADK-RiboJ plasmid through homologous recombination on the basis of gcd-pRADK (Figure 2), and then transformed it into DR. And we verified the length of the recombinant plasmid to ensure the success of the recombinant plasmid through enzyme digestion (Figure 3). Theoretically, the DR containing the gcd-pRADK-RiboJ plasmid will express more GDH, will have a higher catalytic efficiency, a greater decrease in pH, and a greater range of phosphate ring.

Figure 1. Constitution of PGroES-RiboJ-RBS-gcd gene circuits.


Figure 2. Design of GDH-Pro plasmid (gcd-pRADK-RiboJ).


Figure 3. ElectropHoresis of plasmid gcd-pRADK-RiboJ with enzyme digestions.


We cultured DR R1, DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ respectively in TGY liquid medium, and then determined the growth curve. As shown in Figure 4, DR with high GDH expression has a faster growth rate. Then, we cultured DR containing gcd-pRADK-RiboJ and DR containing gcd-pRADK in TGY liquid medium respectively, and measured the pH every 1 hour. The results are shown in the Figure 5. In addition, the DR containing gcd-pRADK-RiboJ and the DR containing gcd-pRADK were cultured in PKO solid medium, and the results of the phosphate ring were observed, as shown in the Figure 6.

Figure 4. The growth curve of DR R1, DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ.


Figure 5. Changes in pH value of TGY liquid medium culturing DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ respectively.


Figure 6. Phosphate ring in PKO solid medium culturing DR containing gcd-pRADK and DR containing gcd-pRADK-RiboJ respectively. Left: DR containing gcd-pRADK; Right: DR containing gcd-pRADK-RiboJ.


Conclusion

We improved BBa_K2717007 with PGroES promoter and RiboJ sequence, and determined the growth curve in DR, the results showed that BBa_K3560006 expressed higher GDH content, and DR containing this part grew faster. And the above results show that we have successfully constructed the GDH-Pro system in DR, and compared with Phosphate dissolution systems, the GDH-Pro system has a stronger pH decrease speed and range, and has a stronger ability to dissolve phosphate.

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).