Difference between revisions of "Part:BBa K4182008"

Revision as of 03:39, 14 October 2022

AA cluster

AA, aspartic acid, is a novel natural herbicide that can be synthesized by fungi (Yan Y et al, 2018). AA targets dihydroxylation dehydrase (DHAD) in the synthesis pathway branched-chain amino acid and leads to the growth inhibition of plants. Branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine, are essential nutrients for plant growth, and the key point of their biosynthetic pathways are dihydroxydehydrase (DHAD) which catalyzes αβ-dihydroxylation dehydration reaction to form the precursor α-ketoacid. DHAD is highly conserved in different plant species and DHAD with its BCAA biosynthetic pathway does not exist in mammals, making it an ideal target for herbicides. The biosynthetic pathway of AA includes: the precursor pGPP is synthetized via MVA pathway from glucose, which will be catalyzed by FPPS to generate FPP, and eventually to AA by astABC gene cluster. astABC gene cluster was from the soil fungus Aspergillus terreus, and were codon-optimized based on E. coli, chemically synthesized, and cloned in our study. Our primary work on the novel herbicide aspartic acid and its astABC gene cluster will benefit its wide applications in the future.

Sequence and Features

Profile

Base Pairs

Design Notes

This gene cluster has been optimized for E. coli

Source

LacI:E.coli FPPS:Rhodobacter azotoformans fpps gene for farnesyl diphosphate synthase, partial cds(GenBank: AB053174.1)

Usage&Biology

Construction and validation of AA synthetic plasmid (plasmid 3).

FPPS and astABC (from the soil fungus Aspergillus terreus) were codon-optimized and genetically synthesized according to E. coli, respectively. where astAB and astC are present on two separate plasmids, respectively.

The final plasmid III uses the medium-copy plasmid MCS1 as the backbone (to avoid metabolic stress caused by high-copy plasmids), contains the astABC trigene and specific transcription terminator T1 from the E. coli rrnB gene regulated by the lac promoter, and multiple highly active ribosomal binding sites (RBS1-3). The astABC gene, LacI-Plac regulatory sequence, and MCS plasmid skeleton were obtained using PCR technology, respectively, and the final plasmid 3 was obtained by further one-step ligation using the golden gate technique.

Figure 3: The plasmid in which Ast ABC is located

Figure 4: The MCS skeleton used and a brief illustration

Figure 5: Complete plasmid profile finally constructed by the experimental group

Figure 6: Agarose gel electrophoresis of the LacI gene

Figure 7: Colony PCR glue plot of plasmid 3 (the presence of the marker gene is used to prove the presence of the plasmid)

References

[1]Resistance-gene-directed discovery of a natural-product herbicide with a new mode of action