Difference between revisions of "Part:BBa K4182010:Design"

Latest revision as of 07:15, 12 October 2022

Critical synthesis circuitry from important precursor GPP to AA

Profile

Base Pairs

10725

Design Notes

The necessary E.coli. codon optimizations were made.

Source

LacI:E.coli 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)

3. Verification and prediction of the herbicidal activity of plasmid 3 expression products

Due to the long cycle of plant experiments and the limited project time, we did not conduct plant experiments after successfully constructing expression plasmids. In the paper "Resistance gene-directed discovery of a natural-product herbicide with a new mode of action" (Yan Yan et al, 2018) The activity of AA was verified in detail and accurately, and the results showed that 250uM AA can have an efficient effect on killing plants, compared with glyphosate, transgenic plants containing DHAD inhibitor proteins. It has obvious resistance to AA, thus indicating the potential of this herbicide in responding to counter-weeds.

Figure 6: Experimental results of Yan Yan et al. (a, b, c, d show that AA has a significant weeding effect in the actual soil and determine that it is caused by herbicides)

References

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