Part:BBa_K4182010
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
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000COMPATIBLE 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.
Figure 1 The synthetic pathway of AA
2. The construction and verification of the AA synthesis circuit (Plasmid 3)
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).
Figure 3: The astABC gene was synthetized and cloned into two donor plasmids
Figure 4: Figure 4 The map of plasmid 3
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.
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
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