Citrus greening disease, or HLB, is one of the most serious diseases caused by a vector-transmitted pathogen, the Candidatus Liberibacter asiaticus. Recently, an ssDNA phage named CLasMV1 was identified in a C. Las strain in Guangdong via metagenomic analysis [1]. It is anticipated to engineer the phage to suppress the growth of CLas and transmission vector D. citri via antimicrobial peptide (AMP) and delicately designed dsRNA.
However, given the concerns in bio-security and material avaiability, the phage CLasMV1 was not directly utilized. The major capsid protein of CLasMV1 is hence expected to detect C. Las specifically and efficiently. Hence, phagemid, the phage-derived vectors with the replication origin of a plasmid, was selected to construct the capsid protein expression system.
C. Las is unculturable in lab conditions. A common alternative to C. Las is L. crescens BT-1. However, the alternative model organism grows slowly and has harsh cultivation conditions. To meet the large testing need, a test model based on BL21 E. coli was constructed. LdtR is a member of the MarR family of transcriptional regulators, which is specifically found in C. Las [2]. Therefore, it was chosen as the characteristic trigger of the phagemid system. A plasmid based on the backbone of pRSF was designed to construct the LdtR-expressing model. The plasmid has a Kanamycin resistance gene as the marker. A lac operator is involved in the plasmid to control expression. A T7 promoter and a ribosome binding site are at the upstream of the LdtR gene. HA tag gene is linked after the LdtR gene to track the product.
CLasMV1 phagemid contains the CLasMV1 capsid protein. A lac operator is involved in the plasmid to control expression. Two LdtR-specific promoters, ZnuA2 and LdtP, are alternative promoters for the gene . The gene of the pelB signal sequence is linked to that of the capsid protein, aiming at directing the expressed CLasMV1 capsid protein to the periplasm of the host bacteria.
Our group first did the literature review on which antimicrobial peptide (AMP) we would like to choose as the part of phage platform and for improvement. The previous iGEM team (2019 FSU team) [3] included Cecropin B, Apidaecin IB, Melittin, vejovine, and APO5, making a cocktail. However, this method can only kill the bacteria. Some of them do not have high specificity and even have side effects, such as toxicity to plants. In addition, the efficiency and the effect of injecting the cocktail into the trees remain doubts. Further literature review guides us to a newly found antimicrobial peptide, MaSAMP. This AMP cannot be used to kill the pathogen with high specificity but also increase the citrus tolerance to Citrus Greening [4].
After settling down our target AMP, we decided to improve the sterilizing effect of MaSAMP by changing amino acid sequences. We created our own tool to improve MaSAMP and used biophysical parameters as a reference. For further information, please refer to our software page and git deposite. Our synthesis method before the completion of our phage platform. Considering MaSAMP consists of 67 amino acids including two alpha helixes, quite different from AMPs found before, we would like to apply biosynthesis to avoid the expensive fees. Although this AMP has higher specificity, we cannot guarantee that it will not harm host bacteria during growth and synthesis. To reduce or even hide its toxicity to the host, we use the strategy of a digestible fusion protein. To indicate the production and quantify its amount, we added a GFP protein before the sumo tag. GFP units expressed by hosts can also decrease the fusion protein solubility within water, which promotes purification. The isolation and purification depend on the function of His tag. The position of His tag affects how strong the fusion protein binds with Ni beads. Since the existence of His tag on the MaSAMP may have an impact on its sterilizing effect, we designed two sort orders: GFP + His tag + sumo tag +MaSAMP; GFP + sumo tag +MaSAMP + His tag. In case His tag at the N terminal affects the purification efficiency, we added a TEV protease digestion site. To avoid affecting the respective function of each subunit, we got experience of inserting linker sequences between different units from our previous team members [5]. In conclusion, we designed 3 sequences:
1.“AMP”: GFP + linker sequence + sumo tag + linker sequence + MaSAMP + His tag
2.“FAMP”: GFP + linker sequence + His tag + linker sequence + sumo tag + linker sequence + MaSAMP
3.“BAMP”: GFP + linker sequence + sumo tag + linker sequence + MaSAMP + TEV digestion site + His tag
The testing part will be divided into three parts. The first part is to test whether the fusion protein will still kill hosts. Briefly speaking, we will use the fusion protein dissolved in DMSO to test the potential sterilizing effect. The second part is to test whether the sterilizing effect will change if biosynthesized MaSAMP has or does not have His tag attached at the C terminal. To test whether our improvements work, we will use fluorescent staining to measure the antimicrobial ability of MaSAMP mutants, combined with measuring OD.
Asian citrus psyllid Diaphorina citri is the vector for the bacterium Candidatus Liberibacter asiaticus (CLas) that causes Huanglongbing. Once a citrus tree is infected with the bacteria and develops symptoms of Huanglongbing, the tree will usually be considered unrescuable [6]. Therefore, controlling the number of vectors to cut off the spreading of the bacteria and prevent the trees from being infected with Huanglongbing is a widely applied principle in dealing with Huanglongbing. The traditional method to control Asian citrus psyllid is to use chemical pesticides. However, the toxicity of pesticides to humans and the ecosystem is usually a concern when applying them widely.
The method of RNAi as an alternative to pesticides allows a lower off-target effect. The genes selected as the target of RNAi are unique to Asian citrus psyllid, meaning that they tend to have high efficiency in killing Asian citrus psyllid specifically. We also proposed using phage as a platform to carry RNA expression genes to CLas. When D. citri ingest this type of CLas, they will be exposed to the RNA that can silence critical genes in their system and eventually cause the death of the psyllid. Compared to traditional pesticides, RNAi through phage platform features high efficiency and low off-target effect, which we think can be meaningful to the citrus industry.
According to the literature review, sequences from six D. citri genes were picked as our target at first, including muscle protein 20 [7], a transformer 2 homologue [3], a cuticle protein [4], cytochrome P450 [5], a sucrose hydrolase homolog [6], and a carboxyesterase gene [7]. We chose the HT115(DE3) + L4440 system, which was originally used to feed Caenorhabditis elegans to apply RNAi, to test the efficiency of different experiment groups. This strategy has been proven to be effective by previous iGEM Team TEC-CEM 2017 [8]. The L4440 vector contains a pair of IPTG-inducible T7 promoters on both sides of the multiple cloning site, which allows the system to express reverse-complementary RNA fragments once the target DNA fragment is inserted into the multiple cloning site. HT115 E. coli strain is an RNase III-deficient strain, which allows the dsRNA produced to accumulate in the cell. After cell lysis and purification, we expect to obtain a considerable amount of target dsRNA. In addition to a pair of T7 promoters, we constructed a hairpin structure for each of the T7 promoters, expecting a higher productivity of RNA compared to without double hairpin structure. At the same time, for three of the genes, we also designed a version with an additional pair of T7 promoters, expecting it to have a higher productivity than without an additional pair of T7 promoters. Once the strains containing the plasmids are constructed, the productivity of each strain will be tested. After culturing and adjusting the OD value to the same for each sample, the total RNA and dsRNA will be extracted and quantified according to the protocol provided by Delgado-Martín & Velasco, 2021 [10]. The RNAi efficiency will then be tested on D. citri nymphs. After soaking the D. citri nymphs in dsRNA solution, we’ll examine the RNA expression of the target gene immediately as well as check the mortality of the nymphs after culturing them on Murraya exotica trees for another week. The strains with the highest interference efficiency and the highest overall efficiency will be picked out for further design in two directions. The first direction is CLasMV1. The second direction involves further processing dsRNA into siRNA using the production system mentioned in Huang et al., 2013 [11].
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