Difference between revisions of "Part:BBa K4182010"
(→‎Usage&Biology)
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==Usage&Biology==
 
==Usage&Biology==
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1. Introduction to a novel herbicide (AA)
  
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===1. Introduction to AA and Biosynthesis Pathways===
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AA, aspartic acid, is a novel natural herbicide that can be synthesized by fungi (Yan Y et al, 2018). In the situation of increasing tolerance to existing herbicide of glufosinate (APHTHINE), AA offers another environmentally effective and low-tolerance option with significant results (See the results of Yan Y et al). 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 is shown as follows. 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.
  
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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 [https://doi.org/10.1038/s41586-018-0319-4] 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:
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[[File:2-1.png|500px]]
  
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[[File:XJTU-p3-1.png|700px]]
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Figure 1 The synthetic pathway of AA
  
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Figure 1: Precursor synthesis
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2. The construction and verification of the AA synthesis circuit (Plasmid 3)
  
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[[File:XJTU-p3-2.png|500px]]
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[[File:AA2-2.png|400px]]
  
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[[File:XJTU-p3-3.png|500px]]
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Figure 2 The AA synthesis circuit
  
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Figure 2: Biochemical synthesis steps of AA
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fpps and astABC (from the soil fungus Aspergillus terreus) were codon-optimized based on E. coli and chemically synthesized. And the synthetized astAB and astC are cloned into two separate plasmids as shown in Figure 3. In order to avoid the metabolic stress caused by high-copy plasmids, the AA synthesis circuit (Plasmid 3) was constructed based on the medium-copy number backbone pBBRMCS1. It contains the astABC gene cluster regulated by the lac promoter and the specific transcription terminator of E.coli rrnB gene, as well as several high-efficient RBS (RBS1-3) (Figure 3). The astABC gene cluster, LacI-Plac regulatory sequence, and linear pMCS1 plasmid backbone were obtained by PCR respectively, and final plasmid 3 was constructed one-step Golden Gate assembly. The plasmid 3 was confirmed by colony PCR verification and gene sequencing (Figure 4).
  
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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.
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[[File:XJTU-p3-4.png|400px]] [[File:XJTU-p3-5.png|350px]]
  
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===2. Construction and validation of AA synthetic plasmid (plasmid 3). ===
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Figure 3: The astABC gene was synthetized and cloned into two donor plasmids
  
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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.
 
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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.
 
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[[File:XJTU-p3-4.png|400px]]
 
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[[File:XJTU-p3-5.png|350px]]
 
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Figure 3: The plasmid in which Ast ABC is located
 
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[[File:XJTU-p3-6.png|500px]]
 
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Figure 4: The MCS skeleton used and a brief illustration
 
  
 
[[File:XJTU-p3-7.png|500px]]
 
[[File:XJTU-p3-7.png|500px]]
  
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Figure 5: Complete plasmid profile finally constructed by the experimental group
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Figure 4: Figure 4 The map of plasmid 3
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[[File:XJTU-p3-8.png|300px]]
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Figure 6: Agarose gel electrophoresis of the LacI gene
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[[File:AA2-3.png|500px]]
  
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[[File:XJTU-p3-9.png|300px]]
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Figure 5 Fragments used for construction of plasmid 3 and colony PCR verification
  
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Figure 7: Colony PCR glue plot of plasmid 3 (the presence of the marker gene is used to prove the presence of the plasmid)
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3. Verification and prediction of the herbicide activity 
  
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===3. Verification and prediction of the herbicidal activity of plasmid 3 expression products===
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Due to the long cycle of plant experiments and the limited time, we did not conduct plant experiments. However according to 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 extensively studied and showed that 100 μM AA exhibit an efficient activity to kill plants. And transgenic plants containing AA-inhibitor protein DHAD has an obvious resistance to AA, indicating the potential of our herbicide to kill weeds. Our primary study on the novel herbicide will promote its further research and applications in the future.
  
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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.
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[[File:AA2-4.png|500px]]
  
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[[File:XJTU-p3-10.png|500px]]
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Figure 6 Activity test of AA conducted by Yan et al
  
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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)
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==References==
 +
[1]Resistance-gene-directed discovery of a natural-product herbicide with a new mode of action
  
 
==References==
 
==References==
 
[1]Resistance-gene-directed discovery of a natural-product herbicide with a new mode of action
 
[1]Resistance-gene-directed discovery of a natural-product herbicide with a new mode of action

Revision as of 03:59, 14 October 2022


Critical synthesis circuitry from important precursor GPP to AA

Plasmid III uses the medium-copy plasmid MCS1 as the backbone (to avoid metabolic stress caused by high-copy plasmids), including the astABC trigene and a specific transcription terminator T1 from the E. coli rrnB gene regulated by the lac promoter, as well as 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 .

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3626
    Illegal EcoRI site found at 7595
    Illegal EcoRI site found at 10513
    Illegal PstI site found at 5844
    Illegal PstI site found at 6024
    Illegal PstI site found at 7131
    Illegal PstI site found at 8462
    Illegal PstI site found at 10270
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3626
    Illegal EcoRI site found at 7595
    Illegal EcoRI site found at 10513
    Illegal PstI site found at 5844
    Illegal PstI site found at 6024
    Illegal PstI site found at 7131
    Illegal PstI site found at 8462
    Illegal PstI site found at 10270
    Illegal NotI site found at 2615
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3626
    Illegal EcoRI site found at 7595
    Illegal EcoRI site found at 10513
    Illegal BamHI site found at 5902
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3626
    Illegal EcoRI site found at 7595
    Illegal EcoRI site found at 10513
    Illegal PstI site found at 5844
    Illegal PstI site found at 6024
    Illegal PstI site found at 7131
    Illegal PstI site found at 8462
    Illegal PstI site found at 10270
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3626
    Illegal EcoRI site found at 7595
    Illegal EcoRI site found at 10513
    Illegal PstI site found at 5844
    Illegal PstI site found at 6024
    Illegal PstI site found at 7131
    Illegal PstI site found at 8462
    Illegal PstI site found at 10270
    Illegal NgoMIV site found at 343
    Illegal NgoMIV site found at 5610
    Illegal NgoMIV site found at 7733
    Illegal NgoMIV site found at 9063
    Illegal AgeI site found at 183
    Illegal AgeI site found at 7888
    Illegal AgeI site found at 9688
  • 1000
    COMPATIBLE WITH RFC[1000]


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 a novel herbicide (AA)

AA, aspartic acid, is a novel natural herbicide that can be synthesized by fungi (Yan Y et al, 2018). In the situation of increasing tolerance to existing herbicide of glufosinate (APHTHINE), AA offers another environmentally effective and low-tolerance option with significant results (See the results of Yan Y et al). 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 is shown as follows. 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.

2-1.png

Figure 1 The synthetic pathway of AA

2. The construction and verification of the AA synthesis circuit (Plasmid 3)

AA2-2.png

Figure 2 The AA synthesis circuit

fpps and astABC (from the soil fungus Aspergillus terreus) were codon-optimized based on E. coli and chemically synthesized. And the synthetized astAB and astC are cloned into two separate plasmids as shown in Figure 3. In order to avoid the metabolic stress caused by high-copy plasmids, the AA synthesis circuit (Plasmid 3) was constructed based on the medium-copy number backbone pBBRMCS1. It contains the astABC gene cluster regulated by the lac promoter and the specific transcription terminator of E.coli rrnB gene, as well as several high-efficient RBS (RBS1-3) (Figure 3). The astABC gene cluster, LacI-Plac regulatory sequence, and linear pMCS1 plasmid backbone were obtained by PCR respectively, and final plasmid 3 was constructed one-step Golden Gate assembly. The plasmid 3 was confirmed by colony PCR verification and gene sequencing (Figure 4).

XJTU-p3-4.png XJTU-p3-5.png

Figure 3: The astABC gene was synthetized and cloned into two donor plasmids


XJTU-p3-7.png

Figure 4: Figure 4 The map of plasmid 3

AA2-3.png

Figure 5 Fragments used for construction of plasmid 3 and colony PCR verification

3. Verification and prediction of the herbicide activity

Due to the long cycle of plant experiments and the limited time, we did not conduct plant experiments. However according to 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 extensively studied and showed that 100 μM AA exhibit an efficient activity to kill plants. And transgenic plants containing AA-inhibitor protein DHAD has an obvious resistance to AA, indicating the potential of our herbicide to kill weeds. Our primary study on the novel herbicide will promote its further research and applications in the future.

AA2-4.png

Figure 6 Activity test of AA conducted by Yan et al

References

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

References

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