Composite
Part:BBa_K3560004
Designed by: ShiDi Xiao Group: iGEM20_XHD-Wuhan-China (2020-10-23)
PGroES-DrRBS-gcd
The gcd gene of Escherichia coli expresses glucose dehydrogenase (GDH), GDH is a membrane-bound quinone protein that catalyzes the conversion of glucose to gluconic acid, generates hydrogen protons, and decrease the pH value. but it needs to be combined with Pyrroloquinoline quinone (PQQ) coenzyme, then the holoenzyme can function normally. Deinococcus radiodurans constitutively expresses PQQ, which can form holoenzymes with GDH from E. coli to catalyze the production of gluconic acid.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 190
Illegal AgeI site found at 1797 - 1000COMPATIBLE WITH RFC[1000]
Introduction <h2>
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>
1. Deinococcus radiodurans culture:
We cultivated DR in the laboratory, then placed it on a plate and observed the growth process of DR. The results are shown in the figure1. In order to further understand the growth characteristics of DR, we need to determine the growth curve of DR. In order to determine the growth curve of DR, DR was cultured in TGY liquid medium at 28 degrees Celsius and 200rpm in a shaker, and the OD600 value of the bacterial solution was measured by a spectrophotometer. The growth curve of DR is shown in the figure2. Compared with E. coli, DR has a longer growth cycle and takes a long time to culture.
Figure 1 DR streak separation and culture.
Figure 2 The growth curve of DR.
2. Phosphate dissolving system:
As mentioned in our DESIGN page, Phosphate dissolving system has four parts: PGroES(BBa_K3560002), RBS(BBa_K3560003), gcd(BBa_K2717000), TT(BBa_B0015) (figure3). In order to realize the function of gene circuit, we construct the following plasmid gcd-pRADK (figure 4). And we verified the length of the recombinant plasmid to ensure the success of the recombinant plasmid through enzyme digestion (figure 5). Theoretically, after gcd-pRADK is transformed to DR, DR can secrete gluconic acid, gradually decrease the pH of the solution, and promote the dissolution of phosphate. The DR containing gcd-pRADK and wild-type DR were cultured in TGY liquid medium, and the pH was measured every 1 hour, the results are shown in the figure 6. In addition, 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 7.
Figure 3 Constitution of PGroES-RBS-gcd gene circuits.
Figure 4 Design of phosphate dissolving plasmid (gcd-pRADK).
Figure 5 ElectropHoresis of plasmid gcd-pRADK with enzyme digestions.
Figure 6 Changes in pH value of TGY liquid medium culturing D.r R1 and D.r containing gcd-pRADK respectively.
Figure 7 Phosphate ring in PKO solid medium culturing D.r R1 and D.r containing gcd-pRADK respectively.
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