Progress 02/15/19 to 02/14/23
Outputs
Target Audience: 1. Graduate students, undergraduate students, postdoctoral researchers, and scientific staff involved in this project through research and laboratory experiences. 2. Students at all levels via classroom teaching and instruction as well as formal and informal scientific and professional exchange. 3. Colleagues in the materials science and plant research community and other life science areas through the Crop Bioengineering Center website, peer-reviewed publications, conference presentations, and formal or informal scientific and professional exchange. 4. Regulatory and governance experts through invited lectures and symposia. 5. The general public through news releases and invited lectures. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?1. One PhD student (100%), responsible for material evaluation and establish models for understanding the mechanism; 2. One PhD student (50%), responsible for material synthesis and characterization; 3. One PhD student (50%), responsible for plant protoplast system establishment; 4. One undergraduate student (100%), assist in material synthesis and novel material evaluation; 5. One professional staff (50%), responsible for in planta DNA/protein delivery and novel material evaluation How have the results been disseminated to communities of interest?Outreach activities and conference presentation by PD/Co-PDs: Crop Bioengineering Center 2022 Fall meeting, "Why biolistic delivery is so inconsistent?" by Shan Jiang and Connor Thorpe Outreach activities and conference presentation by personnel involved in the project: Science Bound demos and outreach activities. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1. Establish a library of delivery materials for plant cell delivery (100% completed). We obtained ~ 50 commercial amine molecules, synthesized ~ 20 lipid-like molecules and ~ 5 polymers as the initial library for plant cell delivery. The NMR spectra were obtained to verify the structures of synthesized lipid-like molecules. The amine molecules and lipid-like molecules have been tested for DNA delivery efficiency in plant cells, while the polymers have been tested of DNA delivery efficiency with animal cells. Objective 2. Establish protoplast cells for testing the delivery efficiency (100% completed) Progress has made in establishing a protoplast system for the evaluation of delivery efficiency of CRISPR/Cas9 reagents using novel delivery molecules. We have a robust transfection protocol for Nicotiana benthamiana. We also built a dual marker system using red fluorescent protein mCherry and green fluorescent protein GFP. The co-transfection of both CRISPR/Cas9 reagent and mCherry/GFP plasmids will allow us to assess the effectiveness of the novel delivery molecules. Objective 3. Test delivery efficiency using gene gun method (100% completed). A dual barrel gene gun set up and corresponding protocols have been established to improve the reproducibility of the data. It has shown the error bar has been drastically reduced with the new procedure. More than 30 amines and 10 lipid-like molecules have been tested for DNA delivery efficiency in plant cells. The results suggest that a few amines exceeded the efficacy of the conventional spermidine molecule. The experiments were repeated with both onion and Benthamiana cells. Objective 4. Develop new delivery method based on nanoparticles and new device (100% completed). We have studied the drying patterns of nanoparticles and how surfactant molecules may change the particle assembly structures. We also designed a new barrel using 3D printing to replace the conventional stock barrel. The results show drying pattern is critical to the delivery efficiency. And the new barrel design significantly improved the efficacy. Objective 5. Carry out outreach activities (Year 1 of 2; 100% completed). 2019-07, PI Jiang and graduate student Kyle Miller has developed Office of Precollegiate Programs for Talented and Gifted (OPPTAG) summer course on the topic of materials science. We gave the demos to the students and introduced the many material concepts including water solubility and degradation. The OPPTAG was terminated by the university. However, PI. Jiang and graduate students Kyle Miller and Connor Thorpe have been giving demos and lab tours for Science Bound students, who are URM high school students with potential in math and science.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
K. Miller, C. Thorpe, A. Eggenberger, K. Lee, M. Kang, F. Liu, K. Wang*, and S. Jiang*, Identifying Factors that Determine Effectiveness of Delivery Agents in Biolistic Delivery Using a Library of Amine-Containing Molecules. ACS Appl. Bio Mater. 5, 10, 4972 (2022).
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Y. Li, M. Marander, R. Mort, F. Liu, X. Yong*, and S. Jiang*, Who Wins the Race Near the Interface? Stratification of Colloids, Nano-Surfactants and Others. J. Appl. Phys. 132, 110901 (2022)
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Progress 02/15/20 to 02/14/21
Outputs
Target Audience:1. Graduate students, undergraduate students, postdoctoral researchers, and scientific staff involved in this project through research and laboratory experiences. 2. Students at all levels via classroom teaching and instruction as well as formal and informal scientific and professional exchange. 3. Colleagues in the materials science and plant research community and other life science areas through the Crop Bioengineering Center website, peer-reviewed publications, conference presentations, and formal or informal scientific and professional exchange. 4. Regulatory and governance experts through invited lectures and symposia. ?5. The general public through news releases and invited lectures. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?1. One PhD student (100%), responsible for material evaluation and establish models for understanding the mechanism; 2. One PhD student (50%), responsible for material synthesis and characterization; 3. One PhD student (50%), responsible for plant protoplast system establishment; 4. One undergraduate student (100%), assist in material synthesis and novel material evaluation; 5. One professional staff (50%), responsible for in planta DNA/protein delivery and novel material evaluation; How have the results been disseminated to communities of interest?1. January 2020,Prof. Jiang gave a presentation with Crop Bioengineering Center "Improving Gene Delivery to Plant Cells with Material Science and Machine Learning." 2. March 2020, Prof. Jiang chaired virual American Chemical Society meeting symposium and gave presentation on coating technologies. 3.January 22, 2020, Prof Wang gave a guest lecture for an ISU undergraduate course ABE 469, "CRISPR genome-editing as a modern breeding technology: how do we do it?" 4 .February 3, 2020, Prof Wang was a Speaker for Genetic Seed Testing Super Workshop, Iowa State University, Ames, Iowa 5.October 1, 2020, Prof Wang gave a guest lecture for an ISU undergraduate course AGRON 338, CRISPR genome-editing as a modern breeding technology: how do we do it? 6. November 2, 2020, Prof Wang was a Speaker of Iowa State University Seed Science & Technology Virtual Webinar Series, Zoom, Ames, IA (150 people) What do you plan to do during the next reporting period to accomplish the goals?1. We are in the process of publishing the current results. The manuscript has been reviewed well by Scientific Report and is now under revision; 2. We will furhter study the loading and releasing of DNA on Au particles and correlate the data with delivery efficiency. 3. We plan to develop a new machine learning algorithm to predict the performance of library molecules and optimize the model with more data aquired from experiments. 4. We start to work on protein and CRISPR/Cas9 delivery. We will use HPLC to study the loaind and release, as a preliminary screening method. 5. We plan to publish more DNA delivery results using library molecules.
Impacts
What was accomplished under these goals?
Objective 1. Establish a library of delivery materials for plant cell delivery (Year 2 of 3; 80% completed). We obtained ~ 50 commercial amine molecules, synthesized ~ 20 lipid-like molecules and ~ 5 polymers as the initial library for plant cell delivery. The NMR spectra were obtained to verify the structures of synthesized lipid-like molecules. The amine molecules and lipid-like molecules have been tested for DNA delivery efficiency in plant cells, while the polymers have been tested of DNA delivery efficiency with animal cells. Objective 2. Establish protoplast cells for testing the delivery efficiency (Year 2 of 3; 40% completed). Progress has made in establishing a protoplast system for the evaluation of delivery efficiency of CRISPR/Cas9 reagents using novel delivery molecules. We have a robust transfection protocol for Nicotiana benthamiana. We also built a dual marker system using red fluorescent protein mCherry and green fluorescent protein GFP. The co-transfection of both CRISPR/Cas9 reagent and mCherry/GFP plasmids will allow us to assess the effectiveness of the novel delivery molecules. Objective 3. Test delivery efficiency using gene gun method (Year 2 of 3; 90% completed). A dual barrel gene gun set up and corresponding protocols have been established to improve the reproducibility of the data. It has shown the error bar has been drastically reduced with the new procedure. More than 50 amines and 10 lipid-like molecules have been tested for DNA delivery efficiency in plant cells. The results suggest that a few amines exceeded the efficacy of the conventional spermidine molecule. The experiments were repeated with both onion and Benthamiana cells. Objective 4. Develop new delivery method based on nanoparticles (Year 2 of 3; 40% completed). We have studied the loading and release of DNA and proteins loaded onto Au particles using HPLC. These results are correlated to the delivery efficiency. We will utilize this new protocol to further screen and develop new nanoparticles. Objective 5. Carry out outreach activities (Year 2 of 3; 80% completed). 2019-07, PI Jiang and graduate student Kyle Miller has developed new demos for Science Bound students (premier pre-college program to empower Iowa students of color to pursue degrees and careers in STEM fields). We gave the demos to the students and introduced the many material concepts including ealsticity and shape memory.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Y. Li, F. Liu, S. Chen, A. Tsyrenova, K. Miller, E. Olson, R. Mort, D. Palm, C. Xiang, X. Yong, S. Jiang*, "Self-Stratification of Amphiphilic Janus Particles at Coating Surfaces," Mater. Horiz. 7, 2047 (2020).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2021
Citation:
Kyle Miller, Alan L. Eggenberger, Keunsub Lee, Fei Liu, Minjeong Kang, Madison Drent, Andrew Ruba, Tyler Kirscht, Kan Wang*, Shan Jiang*, "An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue," under revision, Scientific Report, 2021
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Progress 02/15/19 to 02/14/20
Outputs
Target Audience:1. Graduate students, undergraduate students, postdoctoral researchers, and scientific staff involved in this project through research and laboratory experiences. 2. Students at all levels via classroom teaching and instruction as well as formal and informal scientific and professional exchange. 3. Colleagues in the materials science and plant research community and other life science areas through the Crop Bioengineering Center website, peer-reviewed publications, conference presentations, and formal or informal scientific and professional exchange. 4. Regulatory and governance experts through invited lectures and symposia. 5. The general public through news releases and invited lectures. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?1. One PhD student (100%), responsible for material evaluation and establish models for understanding the mechanism; 2. One PhD student (50%), responsible for material synthesis and characterization; 3. One PhD student (50%), responsible for plant protoplast system establishment; 4. One undergraduate student (100%), assist in material synthesis and novel material evaluation; 5. One professional staff (50%), responsible for in planta DNA/protein delivery and novel material evaluation; 6. One postdoc research associates (10%), responsible for material fabrication and characterization. How have the results been disseminated to communities of interest?Outreach activities and conference presentation by PD/Co-PDs: 1. January 13, 2019, Wang gave presentation "CRISPR/Cas directed gene mutagenesis and site targeted T-DNA integration" to Plant & Animal Genome XXVII Conference, San Diego, California. 2. February 5, 2019, Wang gave an invited Littleton Franklin-York Lecture entitled "CRISPR genome-editing as a modern breeding technology - How do we do it?" to College of Agriculture, Auburn University, Auburn, Alabama. 3. February 5, 2019, Wang gave an invited speech "Everything you want to know about GMO but did not dare to ask" to 100 senior students at Auburn High School, Auburn, Alabama. 4. April 24, 2019, Wang gave presentation "The CRISPR Challenges: Known unknowns and unknown unknowns" to Drake University Law School Gene Editing and Agriculture Symposium, Des Moines, Iowa. 5. June 20, Jiang gave oral presentation "Ultrathin Biobased Transparent UV-Blocking Coating Enabled by Nanoparticle Assembly" at ACS Colloid & Surface Science Symposium, Atlanta, GA. 6. June 21, 2019, Wang gave presentation "Stewardship and biosafety in transgenic plant research" to Danforth Workshop "Fundamentals & Practice of Agrobacterium Mediated Plant Transformation", St Louis, Missouri. 7. July 5, 2019, Wang gave presentation on "CRISPR/Cas Directed Gene Mutagenesis and Site Targeted T-DNA Integration in Crop Species" to 2nd International Conference "Plant Genome Editing & Genome Engineering", Vienna, Austria. 8. July 8, 2019, Wang gave presentation on "CRISPR/Cas directed gene mutagenesis and site targeted T-DNA integration in crop species" to University of Milan, Milan, Italy. 9. August 19, 2019, Wang gave presentation "Development of an open source maize genotype for genomic research" to Sainsbury laboratory of Cambridge University, Cambridge, UK. 10. October 3, 2019, Wang gave presentation "CRISPR genome-editing as a modern breeding technology: How do we do it?" to 2019 TAMU Genome Editing Symposium, Texas A&M University, College Station, TX. Outreach activities and conference presentation by personnel involved in the project: Classroom activities: - Morgan McCaw (postdocs of PD Wang) and Jacob Zobrist (graduate student of Co-PD Wang) conducted a half-day high school workshop on "Biotechnology, GMO and Genome Editing" to 24 students, Central Campus, Des Moines, Iowa, April 19, 2019, . Oral presentations: - "Applications of CRISPR/Cas9 and Cas12a in plants: gene knockout and targeted T-DNA integration"; Lee, K., Wang, K., 2019 Crop Bioengineering Center Spring Seminar Series, Iowa State University, Ames, Iowa, April 3, 2019 Poster presentations: - "Novel Delivery Systems for Gene Editing in Plant Cells"; Kyle Miller, Alan Eggenberger, Fei Liu, Minjeong Kang, Ayuna Tsyrenova, Yifan Li, Kan Wang, Shan Jiang. 2019 National Association of Plant Breeders Annual Meeting, Pine Mountain, GA, August 25-29, 2019 What do you plan to do during the next reporting period to accomplish the goals?1. The initial screening already led to molecules that have 3-4 times higher delivery efficiency than the commercial spermidine molecules. We plan to follow the lead, and screen even larger library of molecules. 2. We also discovered the evidence that DNA binding can directly affect the gene delivery efficiency. We plan to obtain more data and get better statistics of the correlation and validate the data. 3. We plan to develop a new machine learning algorithm to predict the performance of library molecules. 4. We start to work on protein and CRISPR/Cas9 delivery. We have already tested the fluorescent labelled CRISPR proteins. 5. We are working on a few major manuscripts to capture the most significant results. These are expected to be published within the next reporting period.
Impacts
What was accomplished under these goals?
Objective 1. Establish a library of delivery materials for plant cell delivery (Year 1 of 2; 50% completed). We obtained ~ 40 commercial amine molecules, synthesized ~ 20 lipid-like molecules and ~ 5 polymers as the initial library for plant cell delivery. The NMR spectra were obtained to verify the structures of synthesized lipid-like molecules. The amine molecules and lipid-like molecules have been tested for DNA delivery efficiency in plant cells, while the polymers have been tested of DNA delivery efficiency with animal cells. Objective 2. Establish protoplast cells for testing the delivery efficiency (Year 1 of 2; 30% completed) Progress has made in establishing a protoplast system for the evaluation of delivery efficiency of CRISPR/Cas9 reagents using novel delivery molecules. We have a robust transfection protocol for Nicotiana benthamiana. We also built a dual marker system using red fluorescent protein mCherry and green fluorescent protein GFP. The co-transfection of both CRISPR/Cas9 reagent and mCherry/GFP plasmids will allow us to assess the effectiveness of the novel delivery molecules. Objective 3. Test delivery efficiency using gene gun method (Year 1 of 2; 40% completed). A dual barrel gene gun set up and corresponding protocols have been established to improve the reproducibility of the data. It has shown the error bar has been drastically reduced with the new procedure. More than 30 amines and 10 lipid-like molecules have been tested for DNA delivery efficiency in plant cells. The results suggest that a few amines exceeded the efficacy of the conventional spermidine molecule. The experiments were repeated with both onion and Benthamiana cells. Objective 4. Develop new delivery method based on nanoparticles (Year 1 of 2; 20% completed). We have studied the drying patterns of nanoparticles and how surfactant molecules may change the particle assembly structures. These results are related to the preparation of gene gun samples. The delivery method based on nanoparticles is planned for the 2nd year of the project. Objective 5. Carry out outreach activities (Year 1 of 2; 50% completed). 2019-07, PI Jiang and graduate student Kyle Miller has developed Office of Precollegiate Programs for Talented and Gifted (OPPTAG) summer course on the topic of materials science. We gave the demos to the students and introduced the many material concepts including water solubility and degradation. 2019-09 & 11, PI. Jiang and graduate students Kyle Miller gave demos and lab tour for Science Bound students, who are URM high school students with potential in math and science.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Olson, E.; Li, Y.; Lin, F.-Y.; Miller, A.; Liu, F.; Tsyrenova, A.; Palm, D.; Curtzwiler, G. W.; Vorst, K. L.; Cochran, E.; Jiang, S., Thin Biobased Transparent UV-Blocking Coating Enabled by Nanoparticle Self-Assembly. ACS Applied Materials & Interfaces 2019, 11 (27), 24552-24559.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Tsyrenova, A.; Miller, K.; Yan, J.; Olson, E.; Anthony, S. M.; Jiang, S., Surfactant-Mediated Assembly of Amphiphilic Janus Spheres. Langmuir 2019, 35 (18), 6106-6111.
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