Difference between revisions of "Part:BBa K4182008"

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==Usage&Biology==
 
==Usage&Biology==
  
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===Construction and validation of AA synthetic plasmid (plasmid 3).===
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1. Introduction to a novel herbicide (AA)
  
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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.
+
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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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:2-1.png|500px]]
  
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[[File:XJTU-p3-4.png|400px]]
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Figure 1  The synthetic pathway of AA
  
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[[File:XJTU-p3-5.png|350px]]
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2. The construction and verification of the AA synthesis circuit (Plasmid 3)
  
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Figure 3: The plasmid in which Ast ABC is located
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[[File:AA2-2.png|400px]]
  
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[[File:XJTU-p3-6.png|500px]]
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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).
 +
 
 +
[[File:XJTU-p3-4.png|400px]] [[File:XJTU-p3-5.png|350px]]
 +
 
 +
Figure 3: The astABC gene was synthetized and cloned into two donor plasmids
  
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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:AA2-3.png|500px]]
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 +
Figure 5 Fragments used for construction of plasmid 3 and colony PCR verification
  
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[[File:XJTU-p3-8.png|300px]]
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3. Verification and prediction of the herbicide activity 
  
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Figure 6: Agarose gel electrophoresis of the LacI gene
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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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[[File:XJTU-p3-9.png|300px]]
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[[File:AA2-4.png|500px]]
  
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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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Figure 6 Activity test of AA conducted by Yan et al
  
 
==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

Latest revision as of 03:58, 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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1877
    Illegal EcoRI site found at 4795
    Illegal PstI site found at 126
    Illegal PstI site found at 306
    Illegal PstI site found at 1413
    Illegal PstI site found at 2744
    Illegal PstI site found at 4552
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1877
    Illegal EcoRI site found at 4795
    Illegal PstI site found at 126
    Illegal PstI site found at 306
    Illegal PstI site found at 1413
    Illegal PstI site found at 2744
    Illegal PstI site found at 4552
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1877
    Illegal EcoRI site found at 4795
    Illegal BamHI site found at 184
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1877
    Illegal EcoRI site found at 4795
    Illegal PstI site found at 126
    Illegal PstI site found at 306
    Illegal PstI site found at 1413
    Illegal PstI site found at 2744
    Illegal PstI site found at 4552
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1877
    Illegal EcoRI site found at 4795
    Illegal PstI site found at 126
    Illegal PstI site found at 306
    Illegal PstI site found at 1413
    Illegal PstI site found at 2744
    Illegal PstI site found at 4552
    Illegal NgoMIV site found at 2015
    Illegal NgoMIV site found at 3345
    Illegal AgeI site found at 2170
    Illegal AgeI site found at 3970
  • 1000
    COMPATIBLE WITH RFC[1000]


Profile

Base Pairs

Design Notes

This gene cluster has been optimized for E. coli

Source

soil fungus Aspergillus terreus

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