Part:BBa_K5335019
The plant immune enhancers composed of VDAL, Nlp20 and Bacillus splip
The part is primarily constituted of two distinct classes of plant immune enhancer, namely VDAL and Nlp20, in addition to a Bacillus subtilis protein expression exocytosis signal, designated as Splip. In order to achieve enhanced performance in Bacillus, a Bacillus-specific ribosome binding site has been integrated into the circuit. In a theoretical scenario, the VDAL and Nlp20 will be expressed and secreted into the extracellular environment. Consequently, VDAL will function as a signal to enhance plant immunity, while Nlp20 will trigger the PTI response in plants. In this project, the part is to enhance the plant's immunity to better defend against nematodes.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
The VDAL protein and Nlp20 peptide in this part is used secreted through the Sec pathway and enhance to immunity of plants. VDAL is a immunity enhancer which promotes the ETI in plants from Verticillium dahliae and Nlp20 is a small peptide with 20-amino-acid-resuides that can promotes PTI in plants from Bacillus subtilis [1][2][3].Splip is a Sec secretory pathway signal from Bacillus subtilis[4].
Structure of plasmid
We conduct a expression vector based on pHT254, a shuttle plasmid that can be expressed in both Escherichia coli and Bacillus subtilis. The coding sequence is followed a salicylic-acid-activated promoter. On the purpose of purification, a 6*His-tag is linked to each fused protein. The vector is shown as Figure 1.
Figure 1. The structure of expression vector. The plasmids is based on pHT254, inserting the circuit through homologous recombination.
Characterization
Agarose gel electrophoresis
The preliminary confirmation of the success of the transformation of the vector (Figure 1.) was achieved through the polymerase chain reaction (PCR) of the transformed E.coli DH5α colony. Subsequently, the target B. subtilis 168 colony was identified through the aforementioned method. The positive band in E. coli DH5α is 1512 bp, which is 1075 bp in B. subtilis 168.
Figure 2. Agarose gel electrophoresis validation of PCR results of E.coli DH5α and B.subtilis 168 colonies. The first lane was loaded with DL2000 DNA ladder.Sizes were marked on the image. We chose 2× Magic Green Taq SuperMix.It contains DNA Polymerase, dNTP, and an loading buffer system, which allows amplification by adding only primers and templates,the product can be electrophoretic directly after PCR,simplify the experimental process and improve the repeatability of the results. The PCR reaction consisted of 15 μL 2 × Magic Green Taq SuperMix, 1.5 μL forward primer (3.75 mM), 1.5 μL reverse primer (3.75 mM), 11 μL H2O and 1 μL colony. (A) M: DL2,000 DNA Marker; 1-6: Colony PCR bands for E.coli DH5α. The target bands are between 2000bp and 1000 bp; (B) M: DL10,000 DNA Marker; 1-6: Colony PCR bands for B.subtilis 168. The target bands are approximately in a line with 1000 bp, the 5 and 6 bands with wrong position could be caused by too much sample volume.
SDS-polyacrylamide gel electrophoresis
The function of VDAL is been confirmed with Arabidopsis thaliana leaves in BBa_K5335025. We further investigate its expression in B.subtilis 168. The result( Figure 3.) demonstrated that Splip-Nlp20-6*His+Splip-VDAL-6*His (pHT254) transferred into the plasmid was markedly expressed in the engineered Bs168 relative to the non-engineered type. The SDS analysis is based on the whole protein from the bacteria body, so that Splip is not spliced and the total weight of recombinant VDAL is 36.4 kDa. However, the expression of Nlp20 (Mr=7.1 kDa) was not examined by SDS-PAGE due to the insufficient size. Though the correct vector has been constructed, the purification method of these effectors from the culture is still waiting for improvement due to the low purifying yield upon to now.
Figure 3. The SDS-PAGE analysis of whole proteins of non-engineered Bs168 and Bs168 bacteriophage carrying expression vectors. M: Prestained Protein Ladder; WT: the non-engineered of B.subtilis 168; NV-1 and NV-2: the 2 loaded samples from the B.subtilis 168 containing the express vector. The target bands are between 35 kDa and 55 kDa, which are highlighted with black arrows.
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