Difference between revisions of "Part:BBa K4182005"

Latest revision as of 04:15, 14 October 2022

FPPs

We designed the upstream control element of the chimeric VVD-AraC fusion structure and the downstream element to verify the effect of the modified operon. We selected sfGFP as the verification protein to efficiently test the expression of the element. The constructed circuit diagram is shown in the following figure.

Sequence and Features

Profile

Base Pairs

413

Design Notes

The codon of E. coli was optimized

Source

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

Usage&Biology

1. Introduction to AA and Biosynthesis Pathways

AA: aspartic acid is a new type of natural herbicide that can be synthesized by fungi, and in today's increasingly increasing tolerance of weeds to existing herbicides of glufosinate (APHTHINE), AA offers another environmentally effective and low-tolerance option with significant results [1] AA targets dihydroxylation dehydrase (DHAD) in the branched-chain amino acid synthesis pathway. Branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine, are nutrients essential for plant growth, and their biosynthetic pathways are key to dihydroxydehydrase (DHAD). DHAD catalyzes α in the BCAA pathway β-dihydroxylation dehydration to form leucine, isoleucine, precursors of valine, and α-ketoacid. And this enzyme DHAD, which is involved in the synthesis of essential amino acids in plants, is highly conserved in different plant species, and even in plants at the far end of evolution, there is still 80% homology. The BCAA biosynthetic pathway does not exist in mammals, and they rely on food to ingest these three essential amino acids, so DHAD is considered an ideal target for broad-spectrum herbicides, and the AA we use here inhibits plant growth by targeting dihydroxylation-dehydase (DHAD) in the plant branched-chain amino acid synthesis pathway (BCAA). Its biosynthetic path is shown in the following figure:

Figure 1: Precursor synthesis

Figure 2: Biochemical synthesis steps of AA

Glucose is synthesized by the MVA pathway to the precursor pGPP (plasmid 2), which in turn is passed through FPP synthetase (FPPS) to obtain FPP, and finally, AA is synthesized by AstABC.

2. 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