This year, we have summitted some documents from experiments or literature to several different parts, providing several new parts to iGEM community.

KillerRed

We have codon optimized KillerRed to play a photosuicide role in Bacillus subtilis. We proved through experiments that KillerRed has a very obvious killing activity in Bacillus subtilis, and this effect is mainly caused by green light excitation. Figure 1. Bacterial OD₆₀₀. change in different colors of light.
The color of the histogram corresponds to the color of the light received by the engineered bacteria, and the color corresponds to the size of the wavelength. Obviously, the engineered bacteria were most sensitive to green light, and the effect decreased with the increase and decrease of wavelength.

Figure 2. Bacterial OD₆₀₀ change over illumination time.
The Control group was treated with Light avoidance, the Green Light group was treated with artificial green light, and the Natural Light group was treated with natural light for a total of 3 hours.

We also tried to point mutate KillerRed by changing its chromophore to change the wavelength of the activating light or increase the killing activity [1]. We constructed nine mutants that mainly changed the first two bases of the chromophores QYG, but unfortunately all of them lost color and had reduced phototoxicity, suggesting that the modification of KillerRed could require multiple mutations.

holin and PGHs

We verified holin and peptidoglycan hydrolase(PGHs),which were found from bacteriophage VB_sup-GOe1.They can effectively lyse Bacillus subtilis. These sequences were obtained from NCBI and the gene was synthesized by Genscript.

We bind the gene to the pHT315 plasmid with xylose operon, which induces the lysis of B.subtilis after the gene is induced by D-xylose. Figure 3. D-xylose induced lysis formal-experiment(hypoxia).

The bacterial solution was divided into two parts, one was ordinary B.subtilis (control) and the other was B.subtilis with recombinant plasmid (Treatment), and D-xylose was added as shown in the figure.

For more information you can see here: BBa_K5335013

We have introduced a novel plant immune activator, VDAL, derived from Verticillium dahliae Aspf2-like protein, into the iGEM Part Registry. We constructed VDAL-CPPs (R9-Tag) and VDAL-6*His variants. VDAL can activate Pattern-Triggered Immunity (PTI) extracellularly and induce endogenous Effector-Triggered Immunity (ETI) when expressed intracellularly. We have validated its function using DAB staining based on reactive oxygen species.

Figure 4. DAB staining images of Arabidopsis leaves.

Treated with: A. ddH₂O (control), B. salicylic acid (SA) (100 μM), C. SpyCatcher-VDAL-CPPs protein (50 μg/mL),
D. a combination of SA (100 μM) and SpyCatcher-VDAL-CPPs protein (50 μg/mL),E. VDAL-6*His protein (50 μg/mL),
F. a combination of SA (100 μM) and VDAL-6*His protein (50 μg/mL) .
Figure 5. Images of DAB-based ROS Assay.

For details, refer to the component：BBa_K5335025、BBa_K5335026

Cry6Aa2

We used Cry6Aa2 insecticidal crystalloprotein from Bacillus thuringiensis YBT-1518. Cry6Aa2 has the ability to kill nematodes. When it enters the gut of the nematode, it can trigger the cell necrosis pathway, which leads to the killing of the nematode. We built Cry6Aa2 into our nematode killing module, and we successfully expressed Cry6Aa2 in Escherichia coli. We provide a new insecticidal protein for iGEM parts libary and raise new options for other teams working on biopesticide development Figure 6. Cry6Aa2 protein was successfully expressed. A. SDS-PAGE .B. Western Blot
M:Protein Marker.A:1 and 3 were not induced by 1M IPTG, and 2 and 4 were induced by 1M IPTG.B.:1 was not induced by 1M IPTG,2 was induced by 1M IPTG.
For details, refer to the component：BBa_K5335006

TbrA/B

We used the tbrA/B genes from Bacillus thuringiensis YBT-1518, which are located in the same gene cluster. TbrA encodes an aconite isomerase, which can catalyze the isomerization of cis-aconite to trans-aconite(TAA) [2], and TbrB encodes a tran-saconite transporter which is able to transport TAA from the interior of the cell to the exterior.
The combination of these two proteins enables the engineering of bacteria to synthesise and secrete TAA, thereby facilitating the further killing of nematodes. For other iGEM teams, we provide a module that can be utilised for TAA fermentation or other industrial fermentations.

Figure 7. TAA was detected in the culture fluid supernatant. A. 10 μM TAA standard. B. 100 μM TAA standard. C. Sample 1. D. Sample 2
For details, refer to the component：BBa_K5335034

TapA and GgaA

In this year's project, in the colonization module, we have introduced two new basic parts to the iGEM parts library: TapA(BBa_K5335016) and GgaA(BBa_K5335015), as well as a composite part(BBa_K5335017), which in our experiments this year proved to significantly improve the colonization ability of Bacillus subtilis. We hope that this work will help future projects that are needed to improve the colonization capacity of Bacillus.
Figure 8.Results of dissolving crystal violet A₅₉₀ in different groups.
GT(l) is the engineered bacterium that contains only TapA, GT(h) is the engineered bacterium that contains both GgaA and TapA, Control is the blank control without adding bacteria.
For details, refer to the component：BBa_K5335034

Aiming at nematode control, our project this year selected Virus-like particles (VLPs) as the delivery platform for nematode control functional components. VLPs have significant potential as artificial vaccines and drug delivery systems. We selected MS2 coat protein (MS2 CP) tandem dimer sequences containing SpyTag found in the literature [3]. The protein can self-assemble to form VLP, expose SpyTag sites on the surface, and can specifically bind to functional proteins connected to SpyCatcher to form a functional protein delivery body. The interior can also contain RNA containing specific sequences, which can be used as a transport carrier for gene silencing. Figure 9. Properly assembled MS2 VLP particles.
For more details, please refer to: BBa_K5335003.