Expression of ZnuA was examined using two recombinant Pichia pastoris strains (GS115 and X-33). The expression level of three clones (WT, ‘SP, ’21) was analyzed during intracellular and secreted expression. Low levels of soluble ZnuA were obtained during intracellular expression of the protein in strain X-33, but purification of the His tagged protein by nickel affinity chromatography was unsuccessful due to the Pichia mitochondrial alcohol dehydrogenase isozyme III competitively binding to the nickel resin. As a result, we have designed multiple new ZnuA fusion proteins that will be used for purification by affinity chromatography on resins other than nickel. The new fusion proteins were prepared using all previously tested lengths of ZnuA (WT, ‘SP, ’21, ’28, and ’40), fused to three separate binding domains (GST, maltose, and cellulose). After sequencing results have confirmed proper gene orientation of the fusion proteins, transformation of Pichia will be carried out by electroporation. The construct expressing the optimal level of active protein will be selected for large scale purification of ZnuA. Assays done using Escherichia coli: ZnuA was expressed in the inclusion body of E. coli. Co-expression of ZnuA with E. coli Spy chaperone protein didn’t improve the solubility of ZnuA. Expression of ZnuA-GST fusion protein was detected in E. coli inclusion body but being process after 2 hours of induction. The HIS6x tag may interfere the folding of ZnuA. Thus, ZnuA genes (WT, .sp, .21, .28, .40) were cloned into other vectors with different tags (pET34b(+): cellulose binding domain. pMAL-c2X: maltose binding protein) for further analysis. Transcription factors: CLIBASIA_02325 (RirA, Rrf2 family, iron responsive) was purified as pure recombinant proteins (0.26 mg/mL, 6mL) in E. coli after adjusting the purification step. Thermal kinetics pre-screening was attempted immediately after the protein was purified, which indicated that this screening method is not suitable for RirA. In order to evaluate the effect of the positive hits from the thermal melting screening on the DNA binding activity of the MarR, CLIBASIA_01180, electrophoretic mobility shift assays (EMSAs) were performed. First, we selected an appropriate DNA fragment containing the putative promoters of both CLIBASIA_01180 and the divergently transcribed CLIBASIA_01185, coding for a delta aminolevulinic acid dehydratase. CLIBASIA_01180 was able bind this fragment as well as the optimal in vitro conditions for complex formation. The next step was to use the three most significant hits from the thermal melting screening, some less significant hits, and the substrate of the delta aminolevulinic acid dehydratase, aminolevulinic acid. This substrate was chosen because the substrate of an enzyme may be involved in regulating the enzyme’s expression. One of the small molecules disrupted complex formation at very low concentrations, whereas the other hits disrupted the complex only at higher concentrations. Aminolevulinic acid did not appear to stabilize or disrupt the complex, and this led us to hypothesize that CLIBASIA_01180 may not be regulating delta aminolevulinic acid dehydratase. Thus, we further examined the genomic context of CLIBASIA_01180 and found that the region upstream of the MarR is also well conserved in diverse species. This region contains genes involved in flavin biosynthesis (ribE and ribA) and amino acid metabolism (glyA), and if CLIBASIA_01180 is regulating these genes, disruption of this regulator would have deleterious effects on Ca. Liberibacter asiaticus.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. The reimbursable agreement with CRDF was established on September 5, 2012. Efforts have been underway during this quarter to recruit a visiting scientist to work on this project. We continue to study the literature to identify vectors to use for a future scFv library made as part of this project. The goal is to find a suitable vector that is not encumbered by intellectual property and patent issues. We are also optimizing the cloning strategies that will be used to move already selected scFv into transgenic plants. We have recently identified a suitable candidate to be a visiting scientist and the necessary arrangements are underway. We have also contacted a laboratory in Germany about the availability of a promising vector developed there. Related research with the existing scFv is underway on project 551.
The objective of this project is 1) to complete the Las genome sequence and conduct comparative genomics studies on the Liberibacter species; 2) to explore the potential role of the microbial community and genetic diversity of Las bacteria in HLB development; 3) to confirm if Las bacteria are seed-transmissible and their role in HLB development. A complete circular genome of Candidatus Liberibacter asiaticus (Las) was obtained using a metagenomics approach and published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin at the USDA-ARS in Parlier, California, we have obtained and published a complete genome sequence of Ca. L. psyllaurous with ca.1.25Mb. All BAC clones of Las were sequenced, and sequence analyses revealed a potential mechanism of GENOME REDUCTION. Based on the variations within the Las prophages, eight different populations (genotypes) have been identified, and their correlations with phenotypes and disease severity of HLB is under investigations. We have characterized the ATP translocase from Las and proved its function using a heterologous E. coli system. This data was published in J. Bacteriol. 192:834-840, 2010. We are currently developing an antibody-based “drug” to target this protein, aimed at disrupting ATP import, which may be important for its survival. We have also characterized the individual genes of two putative zinc operons in Las, confirming only one operon responsible for zinc uptake. Seed transmission of Las was tested in grapefruit, sweet orange, sour orange and trifoliate orange. Relatively high titers of Las were detected from both seed coats and inner seed coats collected from HLB-affected citrus plants. A very low titer of Las was detected from the embryos and seedlings using nested PCR and real-time PCR. Most, if not all the seedlings did not show typical HLB symptoms and contained a relatively low Las bacterial titer for HLB, even in the three to four year old seedlings. The results indicated that the seed-transmitted Las could not cause typical HLB disease by themselves, which suggested “Detection of Candidatus Liberibacter asiaticus was NOT necessarily equal to the presence of “HLB disease” in plants.” Psyllid transmission study on the Las-positive seedlings was performed. High percentage of psyllids acquired Las bacterium but did not have the same bacterial levels as those from HLB-affected citrus plants. However, it is FIRST TIME that ONE SEED-TRANSMITTED HLB SEEDLING was confirmed by PCR using several Las-specific primer sets. Graft transmission of the cutting from this HLB plant confirmed this seed-transmitted HLB. Progress on culture of Las bacterium in vitro has been made. Up to 1,000,000 to 100,000,00 cells/ml were obtained within 48 hrs based on qPCR estimation. The Las cells number in the cultures were staggering and decline when they reached to Ct value 22.00 (16S rDNA-based RT-PCR) in liquid media. We are looking into factors affecting further growth. Fifty-one BAC clones with overlapped Wolbachia endosymbiont of Diaphorina citri (wDia) genome sequences were screened using wDia specific primers from BamHI BAC library and were sequenced. The average size for the 51 clones was 85.4kb with 95-100% coverage and the average GC content is 34.3%. Assembly results indicated that due to large amount of repeat elements, such as transposase, only 13 BAC clones were able assembled into 1 scaffold. We are conducting the gap closing for each BAC clone and hope to get the full wDia genome soon.
The objective of this project is to characterize the hypI (renamed as hyvI) gene and determine its effects on insect transmission and/or virulence in host plants. Transient expression with alternative expression systems and RT-qPCR, etc., will be used to elucidate the function of the hypI (hyvII) gene of Las and shed light on the molecular mechanism of this “phase variation” phenomenon; thereby developing a novel control strategy for citrus HLB. In addition, antibodies and probes along with standardized protocols developed during this project can be applied for better detection and differentiation of the HLB bacteria. The hyvI and hyvII within two Las prophages were further characterized and some of the results were published in Applied Environmental Microbiology 77:6663-6673, 2011. “Diversity and Plasticity of the Intracellular Plant Pathogen and Insect Symbiont “Candidatus Liberibacter asiaticus” as Revealed by Hypervariable Prophage Genes with Intragenic Tandem Repeats”. We have developed an improved real-time PCR using SYBR Green 1 (LJ900fr) and TaqMan’ (LJ900fpr) protocols with primers and probe targeting the nearly identical tandem repeats of 100bp hyvI and hyvII. The results were published in Molecular and Cellular probes, doi:10.1016/j.mcp.2011.12.001. Monoclonal antibodies against the partial HyvI protein (only one repeat) were generated, and their sensitivity and specificity were valuated for the detection of HyvI protein expressed in E. coli and HLB-infected citrus and psyllids. All antibodies were able to recognize the E. coli expressed HyvI antigen, but were not able to detect the HyvI antigen from HLB-infected plants and psyllids. To determine the cellular localization of the HyvI protein in plant cells and the role of the two putative NLSs in hyvI gene, full-length hyvI and C-terminal region including two putative NLSs were amplified and cloned into pCX-DG vector with GFP driven by CaMV35S promoter. The results indicate that the HyvI protein did not target in plant nucleus but located in cytoplasm (possible in organelle) when transient expression in tobacco. RT(Reverse Transcriptase) PCR confirmed the hyvI gene expression both in the host plant and psyllids though the HyvI protein was not easily detected with Western blot. When the full length (12 full repeats) of hyvI gene was cloned into pUF047plasmid, and replicated in heterologous hosts, the repeat number of hyvI gene remained the same in E. coli, but varied in Xanthomonas citri (citrus canker bacterium), ranging from 1.0 to 10.0 full repeats. Clones of X. citri containing the hyvI gene displayed different degree of growth retardation, indicating potential toxic effect of hyvI gene to X. citri. HyvI C- terminus and full length HyvII was fused to GFP and expressed in E. coli driven by T7 promoter. Confocal microscopy results show both proteins are localized to the bacterial poles. Protein localization, sequence analysis and protein structure prediction suggest both protein belong to autotransporter family. The protein was determined to localize at cell surface by dot blot, and furthermore the protein surface localization was proved by protease treatment of intact bacterial cells. Immunofluorescence assay is underway to confirm the surface localization of the HyvI protein.
Volatiles-based HLB detection: We are planning to conclude the yearly study at the CREC facility (Lake Alfred, FL) for the collection of SPMEs, Twisters (GC/MS) and DMS data on HLB infected and healthy control trees with the last trip in January, 2012. As it was determined that sampled orchard trees are co-infected with CTV, a parallel small-scale study is carried out using CTV-free greenhouse seedling trees at CREC. Also, PCR testing was conducted for orchard trees used in the study to confirm healthy/infected status. The data analysis of all of the collected data is ongoing. For Twister GC/MS studies spanning two time points few months apart, a set of 16 discriminating biomarkers compounds was compiled; 11 compounds were chemically identified. Systematic accuracy for diseased vs. healthy trees discrimination using these biomarkers exceeded 80%. In addition, the large volume leaf volatile purge and trap sample collection procedure has been modified to improve reproducibility. Approximately 60 volatiles have been monitored on consecutive days in recent months. All sampling for both healthy and HLB trees has been taken from the Kinsey block at the CREC. The data is still be analyzed, but initial findings indicated there was a late eluting peak that appears to be a marker for HLB infection even though the tree is non-symptomatic. There was also an early eluting peak (LRI = 948) that was exclusively and consistently found only in apparently healthy trees. This compound has been tentatively identified as pentanal. Sampling in other groves and non-Valencia trees will be carried out to see if these trends continue. Aerial HLB detection: Different detection algorithms were tested including decision tree, modified isomap, shortest distance k-nearest neighbor, and support vector machine. The detection accuracies ranged 56%-78%, and improvements are being investigated. A new set of aerial hyperspectral images were acquired for the CREC and North 40 groves located at the CREC, Lake Alfred, FL on December 14, 2011. Corresponding ground truthing data were also collected during December 13-15, 2011. The images turned out to be of excellent quality with 0% cloud cover. The ground truthing included field spectral measurement, infection status investigation and GPS locations for a total of 386 healthy and infected trees. An 8-band multispectral WorldView-2 satellite images were also acquired for a comparison study. For ground-based remote sensing studies, hyperspectral and thermal data collected using multispectral and thermal cameras are being analyzed. Our research article on fluorescence spectroscopy as a tool for HLB detection has been accepted for publications in ‘Transactions of the ASABE’.
Diagnostic service for growers for detection of Huanglongbing to aid in management decisions, January 2012. As we did in previous reports, this update covers the entire period that Huanglongbing Diagnostic Laboratory has been in service because one of the objectives for the funding is for continued, uninterrupted diagnostic services to growers while expanding our ability to provide diagnostics quickly and assist with research efforts. The HLB Diagnostic Laboratory has been operational at UF-IFAS-SWFREC since February 2008. Since the opening of the lab, there has been continued development of techniques, protocols and efficiency. The lab has been in operation for nearly four years, and as the first week of January, 2012, we have processed nearly 27,000 grower samples, with approximately 600 arriving since the last report, October 2011. Additionally, approximately 19,000 samples have been received for research, of which more than 1900 were within that same time frame. Techniques, Protocols and Research For DNA extractions, we continue to use the magnetic particle based system, which has proved both reliable and fast. Current methods of sample processing have become streamlined and therefore seen little change. We have recently introduced the use of TaqMan Fast Advanced MasterMix for real-time-PCR reactions as this is more economical and has shown comparable-to-superior amplification and detection of gene(s)-of-interest when compared to the TaqMan Fast Universal PCR MasterMix. We now lyophilize all plant samples prior to BeadBeating, which enables superior sample maceration when compared to use of liquid nitrogen. Protocol for the detection of HLB in Asian Citrus Psyllid has been validated, including quantification of HLB in both plant and psyllid samples, with the primary goal of serving research projects within the entomology and plant pathology departments that also contribute funds from their research grants to support the labor and supply costs for research samples. The protocol established in 2010 for the quantification of the HLB bacteria in both the psyllid and host tissue using a standardize curve is being used for research and extension samples. The basic diagnostic service remains available to growers, researchers, extension faculty and dooryard citrus growers. However, we are also expanding the data analysis of PCR-processed-samples to include data from individual groves that consented to have their data used. In conjunction with an epidemiologist and computer mathematician, the spread of the disease will be modeled. These studies are not supported by lab funds but are an offshoot of the database collection. The intent is to have additional tools for looking at the spread of HLB in sites where incidence is still relatively low.
In earlier reports, we have mentioned about superior antibacterial properties and strong retention of copper loaded silica nanogel (CuSiNG) material to plant surface, leading to improved efficacy against citrus canker disease. The objectives are to use less Cu and cut down number of spray applications without compromising protection against the citrus canker disease. To further improve the efficacy of CuSiNG material, we report here the outcome of our recent study on CuSiNG composite material and its antibacterial properties. The CuSiNG composite material has a core-shell nanoparticle design. The core is a pure silica nanoparticle core and the shell is made of CuSiNG material. The purpose of this design is to uniformly spread the active CuSiNG material (the shell layer) on to an inactive pure silica nanoparticle (the core) to take advantage of the high surface area of the nanoparticle core. Our 2011 field trial involves one spray application of the CuSiNG material in every three weeks. At this spray rate, it is expected that a significant amount of Cu in the CuSiNG formulations will remain unused or buried in the film/deposits. In other words, the unused portion of Cu could be potentially cut down from the original formulation without loss of antibacterial efficacy. To test the feasibility of this concept, the core-shell CuSiNG composite materials have been designed and studied. Particle size and the morphology of the core-shell CuSiNG composite material was characterized using transmission and scanning electron microscopy. The thickness of the CuSiNG shell was estimated to be ~ 50 nm. The silica nanoparticle core size was approximately 380 nm. Minimum inhibitory concentration (MIC) of the core-shell CuSiNG composite material was estimated to be 4.9 parts per million (ppm) Cu concentration against both E.coli and B.subtilis. In comparison to Kocide’ 3000 and copper sulfate containing equivalent amount of copper, the core-shell CuSiNG composite material exhibited a 50% and 70% decrease in bacterial growth, respectively. This preliminary study demonstrates that the efficacy of the CuSiNG material could be further improved by having a core-shell design. Note that the size of the silica nanoparticle core and the thickness of the CuSiNG shell are adjustable. This is particularly important as smaller size particles are expected to exhibit high surface area and strong retention properties. In coming reports, we will summarize the outcome of further studies on core-shell CuSiNG composite material. We will also report on the on-going fluorescence imaging studies to understand the interaction of CuSiNG material with bacteria as well as 2011 trial results on CuSiNG material.
In previous reports we have described the preparation of a scFv library prepared in phagemid vector pKM19. The basic scFv library contains 2 x 10_7th unique phage that bind to different antigens present in ‘Ca. Liberibacter asiaticus’ and the psyllid vector. We have also reported that we have isolated scFv from this library that bind to epitopes contained in proteins of ‘Ca. Liberibacter asiaticus’ that are likely to be related to host pathogen interactions and virulence. These epitopes are found on two flagellar proteins, the major outer membrane protein, a pilus protein, a protein believed to polymerize the capsular polysaccharide surface layer of the bacterium, the TolC protein required for survival in a plant host, and InvA, the invasiveness protein that prevents an infected cell from undergoing programmed cell death by apoptosis. The vector and expression system that we have used for this project allows selection of the scFv antibody expressed from a phagemid genome but packaged on the surface of an M13 particle. This facilitates selection, but large scale expression of the scFv requires cloning of the scFv gene into a cognate plasmid expression vector. We have had problems with many of our scFv at this step, because ‘stop’ codons can accumulate in the phagemid without affecting the selection process, but prevent expression from the plasmid vector. During this reporting period we have repaired the improper ‘stop’ codons for several of our scFv to allow full expression of the scFv. These scFv were selected because they showed the desired specificity when selected in the phagemid format, but failed to produce scFv when used in the expression vector. The repairs were done by sequencing the defective scFv and identifying the stop codons, designing primers for a series of PCR that allowed amplification of the scFv while replacing the incorrect codons with corrected sequence, and transforming the corrected plasmid with the scFv into E. coli for expression. We have corrected the sequences for scFv that bind FlhA (scFv B947, B1096, B1072); KpsA (B520, B1199, B1202); the major outer membrane protein OmpA (B743); Pilus protein (B556, B557). These scFv are now available in pKM16, our expression vector for testing. The next steps will require expression of the scFv, purification of the scFv and testing it for yield and specificity against purified antigens. These scFv will be added to our inventory of multiple scFv for each target of interest to improve our chances of finding scFv that will be extremely useful for detection assays and for labeling cells for scientific studies.
We continued working on the identification of sequencing variation within virulence-related genes and development of gene-based phenotyping markers for ‘Candidatus Liberibacter asiaticus’ (CLas). Previously, we selected four groups of virulent genes based on their putative functions in CLas genome. Sequencing analysis found that there were 1-3 single nucleotide polymorphisms (SNP) per gene locus when sequences were compared among HLB strains collected from US Florida, Brazil, China and India. BLASTx analysis indicated that there were two types of nucleotide variations; synonymous and non-synonymous variations. The former is defined as a silent substitution as such changes in nucleotide sequences will not alternate amino acid sequences due to the degeneracy of amino acid codons. Non-synonymous mutations, on the other hand, will result in change in amino acid sequences, thus would likely affect the function of the genes. Bacterial evolution and selection by specific host environments leading up to adaptation and speciation are a cumulative effect of gene loss and gain events. Sequence divergence in coding regions due to mutation accumulation in the individual genes could affect evolutionary fitness and pathogenic characteristics. To extend research, more virulence-related genes were investigated which included; Group 1, heat shock-related protein. In this group, DnaJ and DnaK chaperone proteins were cloned and sequenced. It was reported that the DnaK/DnaJ chaperone machinery was required in many bacteria that caused a systemic infection in the host. In Group 2, CLas secretion systems for type I signal peptidase, tolQ protein, integral membrane protein TerC, colicinV production protein and multidrug resistance transporter protein were cloned and sequenced. Genes involved in the transcriptional regulation system were also investigated (Group3). In this group, putative transcription regulator protein and LysR-type transcriptional regulator (LTTR) were selected. LTTR is one of the key players that help bacteria adapt to different environments. A hrg knockout mutant was found to be sensitive to oxidative products of the respiratory burst, specifically to hydrogen peroxide. A strain overexpressing the hrg gene showed a survival advantage over the wild-type Salmonella under hydrogen peroxide-induced stress. Group 4 contains pili and flagellar genes. Pili are essential for host colonization, virulence and pathogenesis for many bacteria. Flagella-dependent motility is widespread throughout prokaryotes and is advantageous when nutrients are limited. Flagella systems can also play an important role in additional processes such as adhesion to substrates, biofilm formation and host invasion in pathogenic bacteria. Seven CLas genes were identified in this group. They are: chemotaxis protein, pilus assembly protein, pilus assembly protein, flagellar basal body L-ring protein, flagellar biosynthesis regulatory protein FlaF and flagellar motor switch protein G, respectively. Finally, outer membrane protein OmpA and MotB were also included in this study. OmpA/MotB or OmpA is required for pathogenesis in many pathogenic bacteria, and can interact with host receptor molecules. MotB and or MotA serves two functions in E. coli; the MotA(4)-MotB(2) complex attaches to the cell wall via MotB to form the stator of the flagellar motor, and the MotA-MotB complex couples the flow of ions across the cell membrane to movement of the rotor. PCR-based gene cloning and sequencing for above selected gene loci are currently in process. We will establish an in vitro heterogous gene expression system to determinate and characterize the functions of these virulence-related genes.
To demonstrate that Candidatus Liberibacter bacterium is the causative agent of Huanglongbing disease (HLB), or greening, Koch’s postulates need to be performed with a pure culture of Liberibacter. We obtained primo-cultures of Ca. Liberibacter asiaticus (LAS) in insect cell cultures used as feeder cells and we improved our medium and growth conditions. From these primo-cultures we selected LAS cultures cured from insect cells and we checked whether LAS was the only bacteria in our cultures with broad-range PCR based on bacterial 16S rDNA. Other bacteria were found in co-culture with LAS (i.e. a Delftia acidovorans or an actinobacteria strain). Enforced vancomycin selection has been applied to get rid of gram positive contaminants, however LAS signal was lost along the selection. New antibiotics to select for LAS are under investigation. We set up a protocol for mechanical inoculation of the LAS culture to healthy citrus. Three independent LAS cultures (several transfers old) were inoculated to healthy citrus (2 young plants for each CIV). After 5 weeks of inoculation, the 6 inoculated plants were tested by PCR (on 5 leaves). Two plants, inoculated with two different bacterial cultures gave positive PCR detection of LAS. No HLB symptom has been observed to this day (9 weeks after inoculation). We will keep following the evolution of LAS signal in the inoculated trees and check for the onset of symptoms. Inoculations with different LAS CIV will be repeated. Another way to inoculate the LAS culture would be by acquisition through membrane by the insect vector and inoculation of healthy citrus via the infected insect. Experiments of acquisition through membranes are under investigation: psyllids starvation phase followed by a feeding phase of psyllids with the bacteria solution and finally a psyllid/citrus contact phase. Hopefully we will be able to inoculate soon a LAS pure culture to healthy citrus after elimination of contaminant bacteria.
To demonstrate that Candidatus Liberibacter bacterium is the causative agent of Huanglongbing disease (HLB), or greening, Koch’s postulates need to be performed with a pure culture of Liberibacter. We obtained primo-cultures of Ca. Liberibacter asiaticus (LAS) in insect cell cultures used as feeder cells and we improved our medium and growth conditions. From these primo-cultures we selected LAS cultures cured from insect cells and we checked whether LAS was the only bacteria in our cultures with broad-range PCR based on bacterial 16S rDNA. Other bacteria were found in co-culture with LAS (i.e. a Delftia acidovorans or an actinobacteria strain). Enforced vancomycin selection has been applied to get rid of gram positive contaminants, however LAS signal was lost along the selection. New antibiotics to select for LAS are under investigation. We set up a protocol for mechanical inoculation of the LAS culture to healthy citrus. Three independent LAS cultures (several transfers old) were inoculated to healthy citrus (2 young plants for each CIV). After 5 weeks of inoculation, the 6 inoculated plants were tested by PCR (on 5 leaves). Two plants, inoculated with two different bacterial cultures gave positive PCR detection of LAS. No HLB symptom has been observed to this day (9 weeks after inoculation). We will keep following the evolution of LAS signal in the inoculated trees and check for the onset of symptoms. Inoculations with different LAS CIV will be repeated. Another way to inoculate the LAS culture would be by acquisition through membrane by the insect vector and inoculation of healthy citrus via the infected insect. Experiments of acquisition through membranes are under investigation: psyllids starvation phase followed by a feeding phase of psyllids with the bacteria solution and finally a psyllid/citrus contact phase. Hopefully we will be able to inoculate soon a LAS pure culture to healthy citrus after elimination of contaminant bacteria.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. The reimbursable agreement with CRDF was established on September 5, 2012. Efforts have been underway during this quarter to recruit a visiting scientist to work on this project. No suitable candidate has yet been identified. We continue to study the literature to identify vectors to use for a future scFv library made as part of this project. The goal is to find a suitable vector that is not encumbered by intellectual property and patent issues. We are also optimizing the cloning strategies that will be used to move already selected scFv into transgenic plants.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. Permission to use the outside funds was received on September 5. A visiting scientist was hired and started work in October. A very well qualified candidate for the half time student aide job was identified but the hiring process has not yet been completed. Cultures of the clones expressing scFv were revived from storage. Initial efforts have been to improve the yield of scFv expressed from plasmid pKM16 in E. coli. Yields of protein were less than what we had expected. Others working with scFv have had similar problems. Review of the literature provided options to move forward. We have changed the culture medium for the scFv expressing E. coli to a much richer medium and culture at 30C. Differing amounts of IPTG have been tested to better control the induction of expression of the scFv. Yields of scFv proteins have improved. The scFV have been only modestly successful in ELISA, with the absorbance of positive samples always greater than, but usually less than 2X healthy controls. Best results were with scFv to the outer membrane protein of ‘Ca. Liberibacter asiaticus’. Results from dot blots are better, but background absorbance from healthy plant tissue is occasionally a problem. We are currently working on using the scFv in tissue printing assays, because with a low magnification stereomicroscope we can visualize the stain in the phloem tissue to give a better result. During this quarter we have also obtained repairs on our fluorescent light microscope and will use it with fluorescently labeled scFv to detect the pathogen in plant samples.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. There is no research progress to report at this time on the current project. Funding has not been received.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. There is no research progress to report at this time on the current project. The contract was put in place during this quarter and I received permission to use external funds on September 5, 2012. Efforts to recruit a visiting scientist and student aide to perform the research work were made during the summer months. A visiting scientist was identified and the visa and other arrangements were made.
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