Progress 09/01/23 to 08/31/24
Outputs
Target Audience:In nature, microbes play important roles in protecting plants from events such as drought and infection. Occasionally we understand how a microbe is able to help the plant, but often it is not enough to deal with increasingly harsh conditions as climates change. Our ability to engineer microbes has improved substantially in recent years such that we should be able to instruct a microbe to produce exactly what a plant needs when it needs it. We also have new approaches that prevent a microbe from growing unless it is given a special chemical that does not exist in the environment. However, we do not know how long the microbe can survive without the chemical, how much of a beneficial molecule it should make to help protect a crop, and under what conditions if any it can actually help protect this crop. This project uses microbial engineering in the spirit of answering all of these questions with the goal of harnessing biotechnology to safely improve crop yields and to inform risk assessment. During this reporting period, we gained new insights about how a bacterium responsible for plant growth promotion struggles to access inorganic forms of nitrogen under common laboratory incubation conditions (e.g., shaking incubation) but accesses the same forms of nitrogen robustly under static incubation conditions expected at the plant root. We also learned what part of the root of tomato these microbes associate with and how that can be influenced by the provision of certain molecules that the plant or the microbe is capable of producing. We have also designed a system to control where the microbe can survive by making it depend on a synthetic nutrient. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has enabled the training of two graduate students - Avaniek Cabales (in the Kunjapur Lab) and Rachel Warthen (in the Bais Lab). In 2024, Rachel defended her Master's Thesis and graduated from the program. Both Avaniek and Rachel have gotten to attend and present at conferences, including the Synthetic Biology Engineering Evolution and Design (SEED) 2024 and American Society for Microbiology (ASM) Microbe 2024 meetings. The project has also facilitated the hire of a laboratory coordinator at 0.1 FTE to improve maintenance of institutional knowledge. How have the results been disseminated to communities of interest?There have been several talks or poster presentations during this period. See below. Cabales, A., Jones, M., Rosier, A., Warthen, R., Bais, H. and Kunjapur, A., 2024. Towards Engineering Safe Sustainable Agriculture: Engineering and Characterization of Production and Persistence. 2024 Synthetic Biology: Engineering, Evolution & Design (SEED). Cabales, A., Warthen, R. Jones, M., Bais, H., Kunjapur, A., 2024. Towards Engineering B. subtilis for Crop Protection and Persistence. 2024 Chemical and Biomolecular Engineering Winter Research Review. ASM 2024 Conference, American Society for Microbiology 2024 Rachel Warthen, Harsh Bais, Avaniek Cabales, Aditya Kunjapur "Surfactin: Single Handed Hero or Blissful Bystander?" InnovatHER University of Delaware 2024 Rachel Warthen, Harsh Bais, Avaniek Cabales, Aditya Kunjapur "Role of bacterial-derived metabolites in promoting plant and soil health" DENIN Environmental Research Symposium, University of Delaware 2024 Rachel Warthen, Harsh Bais, Avaniek Cabales, Aditya Kunjapur "Role of bacterial-derived metabolites in promoting plant and soil health" What do you plan to do during the next reporting period to accomplish the goals?We are in the final stages of manuscript preparation for one or two papers that relate to the creation of our surfactin producer, the observations about condition-dependent inorganic nitrogen assimilation, the colonization of tomato root by B. subtilis, and the influence of B. subtilis on tomato root characteristics. Besides manuscript writing, our future plans will focus on creating and testing the influence of nsAA-dependent kill switches on biocontained strain persistence (Objective 2), further understanding nitrogen assimilation in static versus shaking conditions (Objective 3), and testing the ability of newly created biocontained UD1022 strains to colonize tomato root only in the presence of supplemented nsAA (a goal related to Objective 4 that is more aligned with the BRAG program as it would provide regulators with quantitative metrics about the performance of these biocontainment systems in the rhizosphere). Objective 1. Complete. Objective 2. OMY dependent degradation in an E. coli kill-switch In E. coli, our lab has constructed a protein degradation system dependent on OMY, such that protein degradation will only occur when OMY is present. We will leverage this system to create a kill-switch, where the presence of OMY results in degradation of a toxic protein. In this case, when the OMY is removed from the system, the protein is left to accumulate and the cell dies. We can layer this with the current OMY dependent E. coli synthetic auxotrophs, which we expect will improve the biocontainment of these strains as layering different containment strategies results in a more stringent biocontainment. Similarly, we can characterize the effects of persistence on these systems. We expect that this system will decrease persistence, as the strains will produce toxins when OMY is removed, which may be faster than the degradation of the auxotrophic marker. We will study the influence of tuning the degradation strengths of the toxin as well as the Ulp1 protease whose expression is required for toxin clearance. Objective 3. Understand and Improve Nitrogen Assimilation We will characterize the conditions under which nitrogen assimilation occurs, namely understanding whether we see this in MS media only. Furthermore, we will engineer gltAB under control of the pHyperspank promoter to determine if we can resolve the poor nitrogen assimilation under shaking conditions. This will also help understand whether dysregulation of nitrogen assimilation results in poor assimilation and we can further probe whether biofilm improves nitrogen assimilation by generating and testing different biofilm knockout strains. Ultimately, this will allow us to better control B. subtilis growth rates and thereby surfactin production. Objective 4. Root Mapping with a Biologically Contained B. subtilis UD1022 strain Now that we know the conditions that permit root association of B. subtilis to certain regions of the tomato root, based on the recent USDA BRAG PI meeting we will connect these investigations to the program goals of understanding biocontainment technologies by studying whether biocontained UD1022 will conditionally colonize tomato root only in the presence of supplemented nsAA. We will use low levels of CFU as our inoculum to try to avoid the occurrence of escapees above the level predicted by our assays of escape from biocontainment in monoculture.
Impacts
What was accomplished under these goals?
Objective 1. We determined a baseline for the persistence of non-biologically contained B. subtilis strains in soil, where persistence is here defined as simply the ability to survive under these conditions. We performed a 30-day experiment where we monitored the persistence of the laboratory strain PY79 and the undomesticated plant growth-promoting bacterium UD1022. We see that UD1022, after an initial CFU reduction of 1 order of magnitude at 1 day, remains steady in the soil at a CFU of 10^6; whereas for PY79 CFU in soil drops over 32 days, resulting in a decrease from 10^7 to 10^4 CFU. Objective 2. As a proof-of-concept for how engineered proteolysis could influence persistence of biocontained strains, we built a system in E. coli that will result in nsAA-dependent degradation of any appropriately tagged protein. The system features the protein Ulp1, which is responsible for cleaving the small ubiquitin modifier or SUMO protein domain. We have modified Ulp1 to contain 2 in-frame UAG codons. When co-transformed with an orthogonal translation system, this construct requires nsAA addition for full-length translation of the Ulp1 protein. The target protein that we wish to degrade is tagged with an N-terminal SUMO domain followed by a strong synthetic N-degron that we constructed (Arg-RAP2.2). Upon nsAA addition, Ulp1 is made, cleaves off the SUMO domain, exposes the strong N-degron, and results in complete degradation of the reporter protein, which is initially super-folder GFP. We have confirmed this phenomenon through Western blot analysis and through microplate reader fluorescence measurements. In the last phase of our project, we will substitute GFP for a toxin protein such that toxin protein accumulates upon nsAA removal. We will also test different C-terminal degrons on our Ulp1 protein so that Ulp1 produced when nsAA is present immediately turns over when nsAA is removed. Objective 3. We found that incubation conditions can have a substantial effect on the ability of B. subtilis strains to grow and produce surfactin. Specifically, we observed this effect in the context of supplying nutritionally complete media that resembles media used for cultivation of plants under hydroponic conditions. We were able to detect surfactin production under static, room temperature conditions. We found that assimilation of inorganic nitrogen is limited for diverse B. subtilis strains under shaking incubation conditions with our baseline medium of ½ Murashige & Skoog (which contains nitrogen in the forms of ammonium and nitrate) supplemented with 1% sucrose, whereas strains grow robustly under otherwise equivalent static incubation conditions or with organic nitrogen sources, specifically L-glutamate or L-glutamine. Furthermore, we constructed root maps to better understand the colonization of PY79, our engineered surfactin producer described in prior reports, and the undomesticated UD1022; as well as the effects of exogenous surfactin and L-glutamate on root colonization.Root mapping was performed with both the wild type surfactin non-producer and surfactin producing engineered strain. The data shows that root colonization was specific to the mature region of root which was independent of surfactin production. In addition, supplementation of glutamate enhanced root colonization irrespective of surfactin production. Interestingly, exogenous supplementation of glutamate also changed the preferential colonization in roots in the mature region. We performed several experiments to better understand how organic nitrogen sources and incubation conditions influence biomass accumulation, since biomass accumulation directly correlates with surfactin titer and allows us to tune surfactin titers, which is a key goal of this project. We found that concentrations as low as 1 mM L-glutamate gave us comparable results as 10 mM, that L-glutamate and L-glutamine play outsized roles as organic nitrogen sources compared to other L-amino acids, and that this behavior is seen for other B. subtilis strains of 168, W23, and undomesticated UD1022. We have begun looking at transcriptomic data to better understand why B. subtilis under these conditions deviates from the expected ability to utilize inorganic nitrogen regardless of static or shaking conditions. Objective 4. Before we monitor the potential of protection of tomato crops by our engineered Bacillus strains, we looked at root colonization as in prior reports. Given the influence of L-glutamate on Bacillus subtilis growth under shaking incubations, and given the hypothesized protective role of surfactin on plants, we also tested the exogenous supplementation of each or both compounds. Tomato seedlings grown in vitro were inoculated with various bacteria and different regions of roots were imaged for root specific colonization. Tomato roots inoculated with PY79 strain showed no colonization in any root region 72 hours post-inoculation. To observe surfactin's potential role in root association, surfactin (25 µg/mL) was exogenously added to 1/2 MS sucrose to prime plants for 48-hrs prior to the addition of PY79. Surfactin was then left in the media (unwashed) or plants were moved to new fresh media not containing surfactin (washed) at the time of inoculation. Post 72-hrs of inoculation, minimal attachment was observed on both washed and unwashed plants by PY79 in the mature region with unwashed plants having slightly more colonization than washed plants. The data suggests that the exogenously surfactin supplemented roots facilitate subtle root association by a surfactin null strain PY79. In addition, the preferential binding by PY79 in the MR (region of maturation) of the root suggests an unknown conducive signaling favoring MR association by the bacteria. PY79 sfp+ was used to inoculate roots and colonization was imaged post 72-hrs of inoculation, but colonization was not observed. To compare how undomesticated strain colonized the roots, a root map of the undomesticated B. subtilis strain UD1022 was also created. We observed that UD1022 colonized both the mature region, and in some cases the elongation zone. To analyze the role of glutamate in surfactin interplay for root colonization phenotype, tomato roots were treated with glutamate (5 mM). Exogenous treatment of Glu to tomato roots resulted in attachment primarily observed within the mature region of the root in both PY79 and PY79 sfp+ and does not extend to either the zone of elongation nor the root tip. This data suggests that addition of Glu may trigger root specific colonization in the MR independent of surfactin. Having shown that plants treated with exogenous glutamate and inoculated with PY79 and PY79 sfp+ showed similar root colonization phenotype, we evaluated the role of exogenous surfactin in the glutamate-surfactin interplay for the root colonization phenotype. When plants were primed with both glutamate and surfactin prior to inoculating roots with PY79, robust colonization was observed in multiple regions of the roots. This colonization occurs in both washed and unwashed conditions at a concentration of 5 mM of glutamate. Addition of 5 mM glutamate did not alter the plant or microbial growth. Interestingly, plant root cultures that were subjected to wash treatment showed less colonization than that of the unwashed glutamate treatments. Roots treated with glutamate and surfactin together showed root colonization not specific to MR and colonized the entire roots. The data suggests that combination of surfactin and glutamate may override root specific colonization patterns triggered by lone glutamate/surfactin treatment. We have also performed assays of pellicle formation as well as assays of the effect of these microbes on primary and lateral root length.
Publications
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Progress 09/01/22 to 08/31/23
Outputs
Target Audience:Our target audience are academic and industrial scientists who are interested in using engineered microbes to boost crop yields. During this second year of funding we have given oral presentations to academic audiences in settings such as a Gordon Research Conference. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project enables the training of two female graduate students, Avaniek Cabales (Kunjapur lab) and Rachel Warthen (Bais lab). Both are second year graduate students at the University of Delaware in different disciplines of chemical engineering and microbiology, respectively. This interdisciplinary project has helped them gain unique skill sets relevant to the agricultural biotechnology industry. How have the results been disseminated to communities of interest?I gave the following invited oral presentations about this work during the reporting period: "Engineering rhizobacteria for biosynthesis of plant effectors and for containment." 2023 American Society of Plant Biologists, Mid-Atlantic Section and University of Maryland Plant Symposium. May 25, 2023. "Thinking outside the bioreactor: Engineering rhizobacteria for biosynthesis of plant effectors and for containment." Single-cell Approaches in Plant Biology Gordon Research Conference. Aug. 3, 2023. What do you plan to do during the next reporting period to accomplish the goals?Future plans will focus on (1) controlling surfactin production, (2) characterizing persistence of synthetic auxotrophs, (3) constructing a kill-switch to tune persistence, and (4) further mapping root colonization, including as a function of supplemented glutamate. 1. Control over Surfactin Production We will clone and characterize a titratable surfactin producer. That is, we will put the srf operon under the control of pHyperspank, which we expect will allow us to control the surfactin production by varying the concentration of IPTG inducer. We also expect that this will allow us to improve the quantity of surfactin produced. 2. Characterization of Persistence of Current Synthetic Auxotrophs We will use plating and recently developed flow cytometry methods (not described earlier due to character constraints) to characterize the persistence and growth of B. subtilis synthetic auxotrophs. This will shed light on some potential persistence mechanisms that are unique to B. subtilis. For example, B. subtilis can form spores which are not metabolically active, but may allow the cells to persist for long periods of time. However, in this state, the cells are not metabolically active, which may also result in deployed cells being less productive. We can characterize this behavior, and measure the persistence versus death versus sporulation of these cells. 3. Kill-Switch Construction Though we initially planned to harness protein degradation to control persistence, our early experiments have not functioned as we anticipated. Thus, we will explore two contingency plans that may also kill cells more rapidly upon induction. First, we will investigate lysis switches in B. subtilis. Instead of inducing degradation of an essential protein, we will induce lysis of the cells, resulting in cell death. We hypothesize that this will allow us to degrade cells at shorter time scales as compared to degradation. As yet another contingency plan, we will explore the use of small interfering RNA (siRNA), whose formation and activity can occur on fast timescales, can result in faster cell death by sequestering important regulatory proteins that bind RNA or preventing translation of new proteins. 4. Root colonization We will evaluate the concentration-dependent surfactin production and associated carbon source (GLu) for root colonization. In addition, supplementation of GLu with surfactin over producing strains will be attempted for the tritrophic interactions, involving P. syringae in tomato plants.
Impacts
What was accomplished under these goals?
In this reporting period, we found major success in designing and characterizing new synthetic auxotrophs in Bacillus subtilis and in achieving among the highest titers of surfactin production reported in the literature. Here, we uncovered a critical role that the supplementation of glutamate plays in boosting surfactin production. We are also making steady progress in understanding where B. subtilis strains colonize on the roots of tomato. Lastly, we have found that previous protein degradation tools are not working in our hands under the conditions tested. 1. Optimizing Plant Media Composition for Surfactin Production Here, we obtained some of the most interesting findings from this reporting period. We cloned and characterized a PY79 surfactin producer, which we refer to as bAC0002. This strain harbors the sfp gene under the Pveg promoter in the LacA locus on the genome. Our initial tests were in LB and S750 media, where we found that this strain produced 0.42 ± 0.037 g/L in LB media and 0.077 ± 0.05 g/L in S750 media at 24 h batch culture endpoints. This was really encouraging; however, these media are typical conditions for cell growth in labs. We then tested the surfactin production in bAC002 at varying concentrations of glutamate in more typical media for plant-microbe interactions: MSNg, 1/2MS (Murashige and Skoog), and 1/2MS with 1.5% sucrose. The final OD of these samples correlated with the amount of glutamate added, with more glutamate resulting in more growth for concentrations from 0-10 mM. To better align our batch monoculturing experiments with our plant experiments, where plants are inoculated with higher OD (0.2) of bacteria, we next tested MSNg, 1/2MS with sucrose and glutamate, and 1/2MS with sucrose at a higher inoculation ratios than our original 1:100 at time points of 24 and 72 hours. These samples were grown together over 3 days. First, while we saw very little to no growth in samples with 1/2MS+sucrose and MSNg media, we now saw detectable surfactin production in MSNg media at these timepoints. More interestingly, we saw a substantial increase in surfactant production for the ½ MS with sucrose and glutamate sample, with production over 0.8 g/L of surfactin. This is a really consequential finding, as we have now shown how the supplementation of just one amino acid - and one that can be found in the root exudates of plants - can dramatically increase the production of surfactin to among the highest levels ever reported in the literature, if not the highest. Although the high titer required both the high inoculation ratio and supplemented glutamate, we do still observe a significant increase in surfactin titer from adding glutamate even at the lower inoculation ratio. Furthermore, this is a higher titer than when using the high inoculation ratio without supplemented glutamate. To investigate whether other sugars or amino acids could similarly dramatically alter surfactin titers, we tested glucose, sucrose, and different amino acids on production. We inoculated and grew bAC002 at a 1:100 cell to fresh media into 300 μL in 96 well plates. Overall, we found that sucrose versus glucose did not have much difference in production. We also tested different amino acids, to determine if they would improve surfactin production. We tested leucine, isoleucine, valine, and aspartate, but saw that bAC002 was only able to grow with valine added, but did not see significantly more surfactin production. We tested PY79 and UD1022 strains in rich and minimal media to determine if high titers of surfactin production would be inhibitory in either media. We measured growth for up to 24 hours on a plate reader with varying concentrations of surfactin added. Fortunately, we found that up to 0.8 g/L surfactin in minimal (S750) and LB media did not affect the growth of either cell. 2. Implication of surfactin production with media modulation on bacterial root colonization. We worked under a speculation that root colonization by benign microbes is usually triggered by root secretions and the root tip, being the highly metabolically active region, may see more colonization. To this end, we generated several root maps, evaluating the root colonization by B. subtilis PY79. We argued that PY79, which does not metabolically synthesize surfactin, may show a different colonization pattern on tomato roots. When analyzing B. subtilis' ability to protect tomato roots. We looked at the root tip to the first lateral as different root zones for the pattern of colonization. When analyzing PY79 on its own, root attachment was not observed in any root region of the plant. This was observed in both MSNg and ½ MS media. We have shown that media such as MSNg and ½ MS media does not show adequate surfactin production by B. subtilis. In addition, we exogenously added 25 μg/mL of surfactin to the MSNg media with the plants for 48-hrs to prime plants. In some experiments the plants were washed and placed in new media so that there was no remaining surfactin in the media and in other cases the plants were left unwashed with the residual surfactin. In both cases attachment was observed within the mature region of the root. This was surprising,as we show that not only surfactin is needed for colonization but also, a specific amount of surfactin drives root colonization. Surprisingly, against the convention, we saw more root colonization at the mature region of the root compared to the CEZ or the root tip. We also modulated media by adding an extra carbon source in the form of glutamate. Glutamate (GLu) was also used in ½ MS media based on the observation that more surfactin was produced under the 10 mM condition without plants. Plants and different surfactin strains under PY79 background were placed in 5 mM, 10 mM, and 20 mM GLu conditions. Attachment was observed with the bDS0182 PY79 reporter, but not bAC0002 surfactin over-producer. Experiments were also conducted at 50 μM, 75 μM, and 100 μM concentrations of glutamate, but no attachment was observed from either strain. An experiment was also conducted with a higher inoculum of bacteria where 1 mL of 107 cells/mL culture was used to inoculate the plants. In this condition, the attachment of bDS0182 was also observed in MSNg media. Removing the carbon sources in both MSNg and ½ MS, glycerol and sucrose respectively, also failed to result in a change of root attachment. Regardless of treatment, when attachment occurs, it is observed in the Region of Maturation in the tomato system. Bacteria cannot be found in either the Central Elongation Zone or the Root Tip. Experiments are conducted in liquid media in 12-well plates, not solid media. Roots of the plants are submerged in 2 mL of liquid (bacterial culture + liquid media).
Publications
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:Our target audience are academic and industrial scientists who are interested in using engineered microbes to boost crop yields. However, during this first year of funding we have not disseminated any results from our study. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project enables the training of two female graduate students, Avaniek Cabales (Kunjapur lab) and Rachel Warthen (Bais lab). Both are first year graduate students at the University of Delaware in different disciplines of chemical engineering and microbiology, respectively. This interdisciplinary project has helped them gain unique skill sets relevant to the agricultural biotechnology industry. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Future plans will focus on (1) the characterization of persistence of our biologically contained strains, (2) the integration of protein degradation tools, (3) engineering surfactin production in B. subtilis PY79, and (4) analyzing root colonization of B. subtilis and E. coli strains. Persistence: Synthetic auxotrophs have been designed to be reliant on a synthetic nutrient for survival. Upon removal of this nutrient, these cells die. However, they do persist for some time after synthetic nutrient removal. We define the kinetics of cell death for these cells as persistence. In deployed settings, these cells do not have the synthetic nutrient necessary for survival, and as such, characterizing persistence helps us understand how these organisms may behave once deployed. In previous persistence measurements, the persistence of DEP has ranged from under two days to over four days, depending on operator. In order to understand why this occurs we will investigate the role of BipA. We hypothesize that the intracellular BipA concentration upon inoculation into non-permissive media may impact how long the cells survive. Similarly, testing cells that have been grown to stationary (as compared to mid-exponential) phase may have different persistence times due to shifts in metabolism from nutrient availability prior to inoculation in non-permissive media. We will also quantify the effects of initial OD, as this will relate to the effects of dosing. We will test persistence by measuring the number of colony forming units (CFU). We will grow 300uL DEP in a 96-deepwell plate and plate 100uL at different time points on permissive agar, which contains the synthetic nutrient. We will also plate 100uL on non-permissive agar to detect any escape or potential contamination. The number of cells that grow on the plate is the number of colony forming units per plated volume. We will also investigate using flow cytometry as an alternative to plating for CFU. By staining the cells with fluorescent dyes, we can measure the total number of viable cells and the number of dead cells. This will also give information regarding an injured cell population and cell phenotypes. Protein degradation: Predicting that deployed settings will behave more like stationary phase systems, we seek to characterize stationary-phase expression and degradation of our fluorescent reporter. From there, we will expand our efforts to investigate proteolysis pathways that may be more suitable for degrading proteins in the stationary phase and stress response-mediated settings that seem more predictive of deployed scenarios. We will investigate degradation by the Lon-polyP complex, which is reported to be involved with amino acid recycling and persistence during amino acid starvation. We will probe degradation tags that could better destabilize our protein of interest during the stationary phase. Surfactin production: It has been identified that the gene sfp when introduced to B. subtilis is required to produce surfactin. As such, we will integrate sfp into the LacA site in the genome using molecular cloning to make a surfactin positive strain of pY79. pY79 has an intact srf operon, but a non-functional sfp gene. Therefore, we expect that by adding a functional sfp gene, we will be able to make pY79 surfactin-positive. Reported titers with supplemented alkanes have reached the grams/liter scale. We will regulate surfactin production through the introduction of synthetic promoters, seeking to develop strains that can produce various concentrations of surfactin. Root colonization: In the coming months, we will test various B. subtilis and sfp mutants for root colonization using our root mapping assay.
Impacts
What was accomplished under these goals?
Protein Degradation Tool Development We have investigated native E. coli protein degradation systems for use in tuning persistence. We base our studies on a reporter assay that has been designed for study of the N-degron proteolysis pathway. The assay utilizes an mCherry-Ubiquitin-degron-sfGFP cotranslational fusion that is scarlessly cleaved into mCherry-Ubiquitin and degron-sfGFP pieces upon expression of ubiquitin protease Ubp1. We utilize the ratio of conditionally stabilized sfGFP fluorescence to the consistent mCherry fluorescence to monitor degron-mediated protein stability. We initially validated this reporter for monitoring degradation in BL21, DH5a, and MG1655 E. coli strains. We recently screened its performance in the DEP-precursor C321 strain. We utilized known destabilizing degrons, including N-degron pathway substrate DPS and in vitro ClpS binding partner FLFVQEL, and known stabilizing degron SLFVQEL as controls to test our reporter assay. We used a Spectramax i3x plate reader to measure kinetic fluorescence and optical density. This assay is performed by cycling between shaking incubated 200 uL cultures at 37C and measuring their optical density and fluorescence, all within the plate reader. We observe 3.12 fold dynamic range between stabilizing and destabilizing degrons in C321 when Ubp1 and the dual reporter are induced at mid-exponential phase. In subsequent studies, we observed that the expression strength of our reporter was critical in obtaining distinct on/off degradation states. Using a lower expression araC/p15a cassette, we found a series of degrons that could destabilize a protein to indistinguishable levels over basal autofluorescence. When the system was ported to a tet/ColE1 vector, we see that expression overwhelms the degradation machinery, revealing the importance of careful protein stoichiometry management. Recognizing that our proposed system needs to be robustly characterized across different expression levels, we are going to study and tune promoter and RBS strength. We seek to utilize De Novo DNA and its promoter and RBS library generation tools to screen a variety of genetic parts. Additionally, we observe that our system varies with induction time. We observe less degradation and lower dynamic range when cells are induced for Ubp1 expression later in exponential phase and stationary phase. However, as there is less degradation, a greater floor of the dynamic range convolutes this at later time points. Detection method for Surfactin Production In order to confirm surfactin production, we have developed an isocratic RP-HPLC method to test surfactin production and concentration. We have observed distinct peaks corresponding to surfactin standards purchased from Smolecule. The lower detection limit was 0.05 g/L with a 10uL injection volume. This procedure was verified to have no overlapping peaks with spent media of pY79. Mesocosm experiment To characterize the ability of Bacillus to colonize tomato roots, we have established a robust root mapping technique. The plants are grown under aseptic conditions, B. subtilis or E. coli were administered in plants and post 24/48 hpi, roots were micro-graphed for bacterial colonization. We are aiming to elucidate which region of the roots are important for bacterial colonization. To this end, we will investigate further the level of colonization from root tip (highly metabolically active region) to the first lateral root emergence site. We speculate that the degree of colonization by Bacillus subtilis may vary within the root region. We also hypothesize that bacterial metabolites such as surfactin may play a role for root association.
Publications
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