Difference between revisions of "Part:BBa K4653114"

(→‎Plasmid construction)
Line 4: Line 4:
  
 
BvEP is an elicitor protein phosphopentomutase from Bacillus velezensis LJ02 which improves the tomato resistance to B. cinerea. High purity BvEP proteins induces the hypersensitivity response (HR) in Nicotiana tabacum. BvEP reduced the rotting rate and lesion diameter of tomato fruits caused by B. cinerea, and increased the expression of Pattern-triggered Immunity (PTI), Effector-triggered Immunity (ETI), systemic acquired resistance (SAR) related genes, ROS content, SOD and POD activities in tomato fruits, while there was no significant effect on weight loss and VC contents of tomato fruits.  
 
BvEP is an elicitor protein phosphopentomutase from Bacillus velezensis LJ02 which improves the tomato resistance to B. cinerea. High purity BvEP proteins induces the hypersensitivity response (HR) in Nicotiana tabacum. BvEP reduced the rotting rate and lesion diameter of tomato fruits caused by B. cinerea, and increased the expression of Pattern-triggered Immunity (PTI), Effector-triggered Immunity (ETI), systemic acquired resistance (SAR) related genes, ROS content, SOD and POD activities in tomato fruits, while there was no significant effect on weight loss and VC contents of tomato fruits.  
−
We designed Bacillus subtilis WB800 to express BvEP and secrete it into the environment to help tomato resist gray mold. We used pMA5 as a plasmid carrier, strong promoter Veg promoter, RBS, Epr signal peptide, BvEP expression gene, and T7TE terminator.
+
We designed Bacillus subtilis WB800 to express BvEP and secrete it into the environment to help tomato resist gray mold. We used pMA5 as a plasmid carrier, strong promoter Veg promoter, RBS, Epr signal peptide, BvEP expression gene, and 2x fd terminator.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 18:57, 10 October 2023


pMA5 - Veg promoter - SpoVG RBS - Epr - BvEP - 2x fd terminator

BvEP is an elicitor protein phosphopentomutase from Bacillus velezensis LJ02 which improves the tomato resistance to B. cinerea. High purity BvEP proteins induces the hypersensitivity response (HR) in Nicotiana tabacum. BvEP reduced the rotting rate and lesion diameter of tomato fruits caused by B. cinerea, and increased the expression of Pattern-triggered Immunity (PTI), Effector-triggered Immunity (ETI), systemic acquired resistance (SAR) related genes, ROS content, SOD and POD activities in tomato fruits, while there was no significant effect on weight loss and VC contents of tomato fruits. We designed Bacillus subtilis WB800 to express BvEP and secrete it into the environment to help tomato resist gray mold. We used pMA5 as a plasmid carrier, strong promoter Veg promoter, RBS, Epr signal peptide, BvEP expression gene, and 2x fd terminator.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 926
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1414
    Illegal BsaI.rc site found at 1482


Biology

BvEP is an elicitor protein phosphopentomutase from Bacillus velezensis LJ02 which improves the tomato resistance to B. cinerea. High purity BvEP proteins induces the hypersensitivity response (HR) in Nicotiana tabacum. BvEP reduced the rotting rate and lesion diameter of tomato fruits caused by B. cinerea, and increased the expression of Pattern-triggered Immunity (PTI), Effector-triggered Immunity (ETI), systemic acquired resistance (SAR) related genes, ROS content, SOD and POD activities in tomato fruits, while there was no significant effect on weight loss and VC contents of tomato fruits. We designed Bacillus subtilis WB800 to express BvEP and secrete it into the environment to help tomato resist gray mold. We used pMA5 as a plasmid carrier, strong promoter Veg promoter, RBS, Epr signal peptide, BvEP expression gene, and T7TE terminator.

Design

The fragment was amplified from the total DNA of B. Velezensis LJ02 by specific primers. We retrieved the total genome of Bacillus Velez and entered the primer sequence after opening its FASTA file on Snapgene.

Figure 1. The location of the primers provided in the literature amplified on the total genome

The fragments simulated by primers were used as BvEP sequences, which were blasted and showed high homology among the bacillus. However, we found that the fragment amplified by the primer was incomplete and lacked the start codon, so we performed blastp on the incomplete sequence in NCBI to obtain the complete amino acid sequence. Finally, the complete sequence of the protein was obtained on the whole genome.

Figure 2. Blast results of nucleic acid sequences on NCBI

Plasmid construction

We synthesized the nucleic acid fragment of Veg promoter-SpoVG RBS-Epr signal peptide-BvEP-T7TE Terminator and designed homologous arm primers required for seamless cloning. The plan is to link the fragment with plasmid vector pMA5 through seamless cloning, and transfer it to Bacillus subtilis WB800 to verify follow-up experiments.

Figure 3. Recombinant vector construction diagram