Methods and compositions for improving phosphate solubilization
A technology of phosphate and acid phosphatase, applied in botany equipment and methods, applications, biofuels, etc., can solve problems such as insoluble form binding
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Embodiment 1
[0259] Example 1 - Reconstituted expression of phoC1 and phoC2
[0260] In order to improve the solubilization of organophosphate by CI019, several plasmids containing CI019 phoC gene reconstituted expression cassettes were designed and constructed. First, the open reading frames encoding phoCl and phoC2 were codon randomized to remove any internal regulatory sequences (SEQ ID NOs: 3 and 4). Next, three heterologous promoter-RBS components were designed: 1) as previously published containing an engineered constitutive lac promoter and an optimized synthetic ribosome binding site (RBS aggagg) (Vick et al., 2011, SEQ ID NO: 5) parts, 2) parts containing promoters BBa_J23110 and RBS BBa_B0032 (parts.igem.org / Catalog, SEQ ID NOs: 6 and 7) from the Registry of Standard Biological Parts, and 3 ) contains the promoter of the constitutive and highly expressed lpp gene of the Enterobacter sucrose strain and components of RBS (CI006, SEQ ID NO: 8). Finally, constructs containing eac...
Embodiment 2
[0261] Example 2 - Analysis of expression of reconstituted phoC1 and phoC2 in plants
[0262] To assess strain transcriptional activity in plants, 600 g of autoclaved sand was weighed into a 656 mL pot, which was filled with sterile DI H 2 Saturated with O and allowed to drain for 24 hours, at which time corn seeds (DKC 66-40) were sown at a depth of 1 cm. Plants were maintained for 4 weeks under fluorescent light at room temperature averaging 22°C (nighttime) to 26°C (daytime) with a day length of 16 hours. Five days after sowing, 1 ml of the cell suspension was inoculated on the seedlings by spraying directly on the new coleoptiles. The inoculum was prepared from 5 ml of an overnight culture in SOB, which was spin pelleted and resuspended in PBS for final dilution to an OD of 1.0 590 (about 10 9 CFU / ml). Control plants were treated with sterile PBS and each treatment was applied to 5 replicate plants. Plants were watered as needed and fertilized Monday-Wednesday-Frida...
Embodiment 3
[0266] Example 3: Solubilization of Phosphate by Klebsiella mutans strains
[0267] Phosphate solubilization screens were performed on one wild-type (WT) and seven mutant strains of Klebsiella mutans 137. Briefly, the assay involves culturing each strain in the presence of an insoluble form of phosphate, collecting the supernatant of the cultured microorganisms, and performing a colorimetric assay on the supernatant to determine the content of soluble phosphate.
[0268] A list and brief description of each strain tested is provided in Table 1. The sequence of each strain is provided in Table 9.
[0269] Table 1: Description of Klebsiella mutans strains
[0270]
[0271] All strains were streaked out from frozen stocks on super optimal broth (SOB) plates. Plates were incubated overnight at 30°C. For each strain, 3 single colonies from overnight plates were inoculated into 5 mL of liquid SOB. These cultures were grown overnight at 30°C with shaking. Optical density (...
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