Designer Algae Biotechnology Risk Assessment - OLD DOMINION UNIVERSITY RESEARCH FOUNDATION

DESIGNER ALGAE BIOTECHNOLOGY RISK ASSESSMENT

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

COMPLETE

Funding Source

OTHER GRANTS

Reporting Frequency

Annual

Accession No.

1010562

Grant No.

2016-33522-25624

Cumulative Award Amt.

$458,605.00

Proposal No.

2016-06238

Multistate No.

(N/A)

Project Start Date

Sep 1, 2016

Project End Date

Feb 28, 2021

Grant Year

2016

Program Code

[HX]- Biotechnology Risk Assessment

Recipient Organization
OLD DOMINION UNIVERSITY RESEARCH FOUNDATION
4111 MONARCH WY STE 204
NORFOLK,VA 23508

Performing Department
Chemistry & Biochemistry

Non Technical Summary
This proposal addresses the Management Practices to Minimize Environmental Risk of GE Organisms program area. It will answer three important questions in biosafety assurance: Can GE algae with transgenic biofuel-production pathways compete with wild-type organisms? What is the fate of the transgenes in GE algae if exposed to non-transgenic organisms? Is it possible to develop a biosynthetic method to limit the spread of GE alga into environments? We will create GE blue-green algae using DNA constructs with biomolecular tracking tags, special PCR primer sequences, and restriction sites to measure the horizontal gene transfer rate among microorganisms, to assess the fate of transgenes, and to test the survivability of GE organisms, all of which is highly relevant to algal biofuels production, food safety, and animal and human health. We will also test using a highly heat-tolerant organism and the inducible insertion of proton-channels into the cytoplasm membrane to disable cell reproduction as methods to limit the spread of GE organisms into environments. The project will contribute greatly to the long-term goals of better understanding GE biotechnology risks and providing a biosafety-guarded GE algae technology for photosynthetic production of biofuels and bioproducts.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
135	4099	1000	50%
135	4099	1080	25%
135	4099	1100	25%

Knowledge Area
135 - Aquatic and Terrestrial Wildlife;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1100 - Bacteriology; 1000 - Biochemistry and biophysics; 1080 - Genetics;

Keywords

ge organism survivability

biosafety

horizontal gene transfer

transgene stability

Goals / Objectives
Genetically modified algae, as a promising source for biomass-based fuel production, are actively being pursued; however, the potential risks of creating such transgenic algae are largely unknown. The long-term goals of the proposed project are to gain better scientific understanding of potential transgenic algae biotechnology risks and to provide a biosafety-guarded designer algae technology for photobiological production of advanced biofuels and bioproducts from carbon dioxide and water. The proposed project will generate new information to assist Federal regulatory agencies in making science-based decisions about the environmental effects of introducing transgenic algae. The proposed project will quantitatively answer a number of important scientific questions with respect to biosafety assurance: (1) Can algae with genetically engineered (GE) biofuel-production pathways successfully compete with wild-type organisms in the environment? (2) What would be the fate and stability (persistence) of the transgenic algae if they were exposed to non-transgenic organisms? (3) Is it possible to develop a biosafety method to limit or completely stop the spread and persistence of transgenic algae into natural and managed environments?The specific objectives (milestones) to achieve the long-term goals of the project are:· Objective 1 (within Year 1): Create special transgenic organisms to assess survivability and horizontal gene transfer;· Objective 2 (within Year 2): Complete the assessment on the fate and stability of transgenes;· Objective 3 (within Year 3): Complete the biofuel-producing transgenic organism survivability tests and demonstrate a biosafety-guarded transgenic alga.

Project Methods
Objective 1 (within Year 1): Create special transgenic organisms to assess survivability and horizontal gene transfer.Task 1. Synthesize DNA constructs to generate GE microorganisms for measuring horizontal gene transfer (Lee and Greene with a Biochemistry PhD Student). We will design and synthesize a special set of transgenes (DNA constructs) to create a set of genetically engineered (GE) microorganisms from the following model host microorganisms that we can readily transform with the molecular genetic tools available in our laboratory: Synechocystis sp. strain PCC 6803, Synechococcus elongatus PCC7942, T. elongatus BP-1, and E. coli (K-12 strain). In addition, specific restriction enzyme-cutting sites will be included in the designs of various DNA constructs so that they can be used later to generate restriction fragments of the transgenes for assessing their fate and stability in cell cultures.Task 2. Create GE organisms by genetic transformation (Lee with a Synthetic Biology PhD Student and a Biochemistry PhD Student). We will use a highly heat-tolerant cyanobacterium, T. elongatus BP-1 (optimal growth temperature 55°C), as a primary host organism, in addition to commonly-used model cyanobacteria such as Synechococcus elongatus PCC7942 and Synechocystis sp. strain PCC 6803. When necessary, we will analyze the expression of designer genes at mRNA and/or protein levels as well, especially for cases where there may be no observable activity of the GE enzymes. We will perform RT PCR assays of mRNA and/or monitor the expression at the protein level with certain fluorescent linked tags.Task 3. Measure the horizontal gene transfer rate among microorganisms (Lee and Greene with two Biochemistry PhD Students). We will measure the rate of horizontal gene transfer (HGT) among various microorganisms selected from the groups of Synechocystis, Synechococcus, Thermosynechococcus, E. coli, and eukaryotic alga Chlamydomonas reinhardtii. We will periodically take samples of the co-cultured cells and plate them on BG-11 agar plates containing the two antibiotics: tetracycline and chloramphenicol, so that only the cells that possess both tetA and CmR from a possible HGT would be able to survive and form colonies. By analyzing the number of the colonies formed per amount of co-culture sample cells plated as a function of co-culture incubation time, we will calculate the probability of HGT.Objective 2 (within Year 2): Complete the assessment on the fate and stability of transgenes.Task 4. Test the use of a highly heat-tolerant photosynthetic organism as a biosafety feature to limit the spread of GE organisms into the environment (Lee with a Biochemistry PhD Student). We will test the growth of T. elongatus BP-1 with a photobioreactor and assess its survivability in a greenhouse where the temperature is controlled below 30oC for cooler season and above 30oC to mimic hot environmental temperature. Since environmental temperatures may often be above 30oC during the summer, we will also determine how long the 30oC treatment under natural sunlight conditions is necessary to kill the bacteria.Task 5. Create a cell-division-controllable photosynthetic organism to limit gene transfer or outcrossing to sexually compatible organisms (Lee with a Synthetic Biology PhD Student and a Biochemistry PhD Student). We will create a cell-division-controllable designer butanol-producing alga by introducing a designer proton-channel gene into a host eukaryotic alga. The expression of the designer proton-channel gene will be controlled by the Nia1 promoter, which can also be used to control the expression of the biofuel-production-pathway genes. When necessary, Dr. Greene (Co-PD) in collaboration with Dr. Jennifer Poutsma will also use computer simulations to optimize the polypeptide proton-channel design and develop a model to predict the expected effects of the envisioned designer proton-channel.Task 6. Assess the fate and stability of transgenes in designer algae (Greene and Lee with two Biochemistry PhD Students). This task can be performed in parallel with Task 4 by comparatively testing a co-culture of GE Thermosynechococcus and its wild-type parental strain with a pure GE Thermosynechococcus culture and a pure Thermosynechococcus elongatus BP-1 culture. These three liquid cultures will be comparatively tested in three separate photobioreactors. We will assess the fate and stability of the transgenes by tracking the DNA of the transgenes in the cells cultures' genomic DNA as a function of culturing time. That is, we will periodically take samples from each of the three liquid cultures and extract genomic DNA. The DNA will be analyzed with a specific set of restriction enzymes that will generate specific restriction fragments of the transgenes, based on the DNA construct design. By monitoring the specific restriction fragments of the transgenes as a function of culturing time, we will be able to assess the fate and stability of the transgenes in designer algae.Objective 3 (within Year 3): Complete the biofuel-producing GE organism survivability tests and demonstrate a biosafety-guarded transgenic alga.Task 7. Assess the survivability of GE organisms in competition with wild-type cells (Lee with two Biochemistry PhD Students). After the preliminary biofuel-producing GE organism is upgraded with better designed DNA constructs and molecular tagging of the key biofuel-producing pathway enzymes, we will set-up incubations of mixtures of GE organisms with the wild-type (WT) cells. We will assess the survivability of GE Thermosynechococcus in competition with its wild-type by analyzing the co-culture cell populations sampled periodically. The assessment of the GE population that contains the set of transgenes with the KanR selectable marker will be performed by plating the co-culture cells on agar BG-11 culture medium in the presence and absence of kanamycin. By counting the number of the GE colonies per amount of cells plated in the presence of kanamycin as a function of co-culturing time, we will be able to calculate the population of the GE cells relative to the WT population and the total population in the absence of kanamycin. We will test the GE cells relative survivability under both biofuel-producing and non-producing conditions. Biofuel-production will be measured using the techniques of GC x GC/MS. The identity of the biofuel products and metabolites including metabolic intermediates will be verified using the state-of-the-art COSMIC analytic facility at Old Dominion University. Some of the key biofuel-production-pathway enzymes, such as the designer nirA-promoter-controlled 2-Keto Acid Decarboxylase fused with fluorescent tags, will be visualized in live cells using a fluorescent microscope. By analyzing the positive colonies, we will be able to assess and characterize the survivability profile of GE organisms in competition with WT cells.Task 8. Demonstrate a biosafety-guarded transgenic alga for photosynthetic production of biofuel (Lee and Greene with two Biochemistry PhD Students). After a biofuel-producing transgenic alga with a biosafety-guarded feature is created through Tasks 4 and 5, we will characterize the function and stability of the transgenes in relation to photosynthetic biofuel production. Furthermore, we will analyze the stability of the transgenes in the biofuel-producing GE organisms by comparatively measuring the probability of horizontal gene transfer with and without a biosafety-guarded feature, similarly as described in Tasks 3 and 6. We will also analyze the effect of the biosafety-guarded feature on the biofuel production stability and on limiting the spread and persistence of GE organisms into natural and managed environments. The latter will be measured also through the comparative survivability tests of GE organisms with and without a biosafety-guarded feature in competition with WT cells, as described in Task 7.

Progress 09/01/16 to 02/28/21

Outputs
Target Audience:The scientific community, policy makers and the photosynthetic biofuel and bioproduct industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In Task 1 three graduate students gained advanced scientific training in genomic analysis. In Task 2 three graduate students gained advanced scientific training in molecular biology and genetic engineering techniques. In Task 3 three graduate students have gained advanced scientific training in microbiology and molecular biology techniques. Since initiating Task 3, two undergraduates (one biology major and one biochemistry post-baccalaureate) have joined the team. In Task 4, three additional undergraduate students (two biology majors and one chemistry major) gained training in microbiology. The research team is comprised of the following students: Thu Nguyen, Graduate Student; Cherrelle Barnes, Graduate Student; Oumar Sacko, Graduate Student; Megan Hept, Graduate Student; Andriana Chrysovalanti Zourou, Graduate Student; Simirjeet Singh, Graduate Student. In 2021, three PhD dissertations (Oumar Sacko summer 2021, Thu Nguyen fall 2021, and Cherrelle Barnes fall 2021) and one MS thesis (Andriana Chrysovalanti Zourou summer 2021) are expected to be awarded, which are related to this research. How have the results been disseminated to communities of interest?Major results have now been presented in the national conferences and published in the peer-reviewed international journals. Additional results are being prepared for submission to a national conference and for publication in international journals. 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: 100% accomplished. Task 1. 100% complete. Progress: Seven genetic constructs called DNA cassettes were designed to contain genes and regulatory signals for production of biofuels and other products to monitor the behavior of the genetic material inside the cyanobacteria. Outcomes: Seven DNA cassettes were synthesized. Each cassette additionally contains signals such as promoters, termination signals to enable the expression of the foreign genes as well as restriction sites and recombination sites for integration into the genome of the target organisms. The eighth DNA cassette was designed utilizing a commonly used promoter (IPTG) in synthetic biology along with other mentioned components. Impact: The methodology developed forms the foundation for the remaining seven tasks in this research study and establishes an approach for rationale design of GE cyanobacteria for biofuel production, risk assessment and containment. Task 2. 100% complete. Progress: Five genetic constructs were successfully transformed into T. elongatus BP1 and two DNA constructs into Synechocystis 6803. Experimental conditions have been developed to identify the presence of the GE molecular genetic construct inside the organism as a circular piece of DNA called a plasmid (non-integrative transformant) and as an integrated cassette in the cyanobacterial genome (integrated transformant) which means the foreign DNA we designed was inserted into the cyanobacterial genome. Recent experiments at the molecular level have showed gene expression in GE T. elongatus BP1 carrying the isobutanol production cassette. Outcomes: Three DNA constructs, one designed to produce alcohol, one designed to produce butanol, and one designed for isobutanol production (KIVD-ADH, KIVD-BDH, and pB-BP1KY respectively) have been verified for successful insertion and incorporation into the cyanobacteria genome. Quantification of isobutanol production from pB-BP1KY by gas chromatography and mass spectrometry is ongoing. The two new constructs that were synthesized and successfully transformed in Synechocystis sp. strain PCC 6803 contained in one, a methanol-producing gene (MeOH-Strep) and in the other a yellow fluorescence protein as well as a lipase producing gene (YFP-R-Lipase). Impact: The potential integration of cassettes into the genome of the cyanobacteria indicates an additional and heightened level of bio-risk assessment needed by researchers and the USDA. Task 3. 100% complete. Progress: In depth studies with GE T. elongatus BP1 containing the KIVD-BDH, KIVD-ADH and YFP-ST-RLip-Kan constructs were conducted. The condition for growing cyanobacteria and E. coli in co-cultures has been optimized for this study. Studies with these different constructs have completed the goal of examining horizontal gene transfer between GE cyanobacteria T. elongatus BP1 and wild-type E. coli DH5α. Outcomes: Co-culture studies with GE T. elongatus BP1 each containing the KIVD-BDH, KIVD-ADH and YFP-ST-RLip-Kan construct and, wild-type E. coli were conducted and successful. Antibiotic gene in the form of plasmids within GE T. elongatus BP1 was showed to be transferred into wild-type E. coli within 2 days of co-culturing. The use of the initial growth media has enabled initial studies to go forward and highlighted the need for improved mixtures of growth media conditions. Different mixtures were tested and optimized. Mixture of GE T. elongatus BP1 and E. coli at 1:1 ratio in their respective medium allows the transfer of antibiotic resistant gene in this study. Impact: The results indicate that non-integrative transformants (those with free DNA constructs inside the cyanobacteria) pose a biosafety risk because of the rapid transfer and we aim to determine if integrative transformants (those with the DNA constructs inserted into the host genome) undergo less horizontal gene transfer thus potentially directing the development of GE organisms. Objective 2:95% accomplished. Task 4. 100% complete. Progress: The greenhouse on the campus at Old Dominion University was set-up to conduct experiments for this research study. The wild-type T. elongatus BP1 and two GE T. elongatus BP1 were grown in liquid cultures and on solid media. alongside a non-thermophilic cyanobacterial strain (Synechocystis 6803) in our project. Outcomes: The study showed that 2 weeks of exposure to the cool temperature conditions (15.44-25.30 °C) was enough to cause complete death of GE T. elongatus BP1. However, it took 2-4 weeks for the wild-type T. elongatus BP1 cells to die. Impact: This study revealed that the thermophilic feature of the T. elongatus BP1 may be used to serve as an effective biosafety mechanism at a cool temperature between 15.44°C and 25.30 °C, but may not be able to serve as a biosafety mechanism at warmer temperatures. Task 5. 85% complete. This task was redesigned to allow for more extensive assessment of horizontal gene transfer to clinically relevant gram-negative and gram-positive bacteria. The strains studied are E. coli K12, E. coli DH5α, Pseudomonas putida, Staphylococcus epidermis and Bacillus licheniformis. The studies aim to improve our bio-risk assessment of the transformed cyanobacteria BP1-99C. Task 6. 100% complete. Progress: A one-year study to assess the stability of GE T. elongatus BP1-pKA (integrative transformation with KIVD-ADH construct) was conducted by monitoring the presence of the transgenes and expression of the kanamycin resistance gene. Outcomes: After one year, the transgenes appear to still be present within GE T. elongatus BP1-pKA cells. PCR of genomic DNA was performed to amplify the insertion site (location on chromosome where foreign genes are inserted), the kanamycin resistance gene. The PCR of the insertion site gave two distinct bands at 2.3 kb and 6.6 kb, indicating the wild-type (or no gene insertion) and insertion of genes, respectively. While the cultures under antibiotic pressure showed the majority of cells still had the transgenes within the insertion site, the cultures without antibiotic pressure showed a gradual increase in cells that did not have the transgenes within the insertion site. Objective 3:90% accomplished. Task 7. 100% complete. Progress: A competition study was conducted by co-culturing a fixed ratio of GE and wild-type cyanobacteria under various environmental conditions and monitoring the change over time. Outcomes: The results indicated that when incubated at room temperature, the GE Synechocystis 6803 (6803-YFP-R-Lipase) does not have a competitive advantage or disadvantage compared to the wild-type Synechocystis 6803. However, in the greenhouse during the cool season (16 oC-26 oC) the wild-type Synechocystis 6803 showed a slight competitive advantage over the GE Synechocystis 6803 (6803-YFP-R-Lipase). Also, under cool season greenhouse conditions, wild-type Synechocystis 6803 was co-cultured with thermophilic GE T. elongatus BP1 BY20 and, wild-type T. elongatus BP1. The GE and wild-type thermophilic strains were outgrown by the Synechocystis 6803 in less than 3 weeks. Task 8. 80% complete. Progress: This task was redesigned to focus on photobiological production of isobutanol biofuel using Thermosynechococcus elongatus BP1. A set of genes were introduced into the cells and isobutanol was detected. Outcomes: The results indicate that the thermophilic strain of cyanobacteria Thermosynechococcus elongatus BP1 is able to produce biofuel (isobutanol). In combination with results from task 4, it is expected that biofuel production using thermophilic cyanobacteria provides a built-in biosafety feature. These genetically modified cells would not likely to survive if they escaped containment of high temperature which would significantly reduce the risk of gene transfer.

Publications

Type: Journal Articles Status: Published Year Published: 2019 Citation: Nguyen TH, Barnes CL, Agola JP, Sherazi S, Greene LH, Lee JW (2019) Demonstration of horizontal gene transfer from genetically engineered Thermosynechococcus elongatus BP1 to wild-type E. coli DH5?, Gene, 704: 49-58.
Type: Journal Articles Status: Published Year Published: 2019 Citation: Lee JW (2019) Protocol measuring horizontal gene transfer from algae to non-photosynthetic organisms, MethodsX 6:15641574.
Type: Journal Articles Status: Published Year Published: 2020 Citation: Sacko O, Barnes CL, Greene LH, Lee JW (2020) Survivability of Wild-Type and Genetically Engineered Thermosynechococcus elongatus BP1 with Different Temperature Conditions, Applied Biosafety: Journal of ABSA International, 25(2): 104-117.
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bio-risk assessment research on genetically engineered cyanobacteria for sustainable biofuel (2019) Nguyen TH, Barnes CL, Greene LH, Lee JW (2019), Experimental Biology 2019, Orlando, FL Abstract ID: lb301
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Competition study between wild type cyanobacteria Synechococcus elongatus PCC 7942 and plasmid transformant Synechocystis PCC 6803 (2019) Sacko O, Greene LH, Lee JW, American Chemical Society National Meeting (Biochemical Technology) 2019, Orlando, FL Abstract ID: 3126326
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Survivability of genetically engineered Thermosynechococcus elongatus BP1 in different temperature conditions (2019) Sacko O, Barnes CL, Greene LH, Lee JW, American Chemical Society National Meeting (Environmental Chemistry) 2019, Orlando, FL Abstract ID: 3128391
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Assessing the Stability and Expression of Transgenes in Genetically Engineered Cyanobacteria for Biofuel Production (2019) Barnes CL, Lee JW, Greene LH, American Chemical Society National Meeting (Biochemical Technology) 2019, Orlando, FL Abstract ID: 3128212
Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Designer biosynthetic pathways for photosynthetic biofuels and bioproducts: opportunities and challenges, Lee JW, poster presented in the Experimental Biology Conference (April 6-9, 2019) sponsored by American Society for Biochemistry and Molecular Biology in Orlando, FL., Abstract Number:486.1 Published Online:1 April 2019 in The FASEB Journal, https://www.fasebj.org/doi/abs/10.1096/fasebj.2019.33.1_supplement.486.1 Abstract ID: 486.1

Progress 09/01/19 to 08/31/20

Outputs
Target Audience:The scientific community, policy makers and the photosynthetic biofuel and bioproduct industry. Changes/Problems:This project has recently been extended based on the USDA-approved non-cost extension of this project to February 28, 2021. What opportunities for training and professional development has the project provided?In Task 1 three graduate students gained advanced scientific training in genomic analysis. In Task 2 three graduate students gained advanced scientific training in molecular biology and genetic engineering techniques. In Task 3 three graduate students have gained advanced scientific training in microbiology and molecular biology techniques. Since initiating Task 3, two undergraduates (one biology major and one biochemistry post-baccalaureate) have joined the team. In Task 4, three additional undergraduate students (two biology majors and one chemistry major) gained training in microbiology. The research team is comprised of the following students: Thu Nguyen, Graduate Student; Cherrelle Barnes, Graduate Student; Oumar Sacko, Graduate Student; Megan Hept, Graduate Student; Andriana Chrysovalanti Zourou, Graduate Student; Simirjeet Singh, Graduate Student. How have the results been disseminated to communities of interest?Some of the results have now been presented in the national conferences and published in the peer-reviewed international journals. More results are being prepared for submission to a national conference and for publication in international journals. What do you plan to do during the next reporting period to accomplish the goals?In consistence with the project timeline as shown in Table 1 of the original project proposal and with the recently approved non-cost extension of the project to February 28, 2021, we aim to be 100% completed with all three objectives by February 28, 2021. Furthermore, we will present our results at both scientific conferences and through submission to top journals for publication. We aim to publish the results of the fate and stability studies. In 2020 results were presented at a National Conference. This project currently supports the thesis research work of six graduate students (Thu Nguyen, Cherrelle Barnes, Oumar Sacko, Megan Hept, Andriana Chrysovalanti Zourou and Simirjeet Singh). The PI (James Lee) and the Co-PI (Lesley Greene) aim to also present research findings at a scientific conference.

Impacts
What was accomplished under these goals? Objective 1: Create special transgenic organisms to assess survivability and horizontal gene transfer 100% accomplished. Task 1. Synthesize DNA constructs to generate genetically engineered (GE) microorganisms for measuring horizontal gene transfer. 95% complete. Progress: Seven genetic constructs called DNA cassettes were designed to contain genes and regulatory signals for production of biofuels and other products to monitor the behavior of the genetic material inside the cyanobacteria. This year, the eighth DNA construct was designed and synthesized for production of isobutanol which is a high potential biofuel source. Outcomes: Seven DNA cassettes were synthesized. Each cassette additionally contains signals such as promoters, termination signals to enable the expression of the foreign genes as well as restriction sites and recombination sites for integration into the genome of the target organisms. The eighth DNA cassette was designed utilizing a commonly used promoter (IPTG) in synthetic biology along with other mentioned components. Impact: The methodology developed forms the foundation for the remaining seven tasks in this research study and establishes an approach for rationale design of GE cyanobacteria for biofuel production, risk assessment and containment. Task 2. Create GE organisms by genetic transformation. 100% complete. Progress: Five genetic constructs were successfully transformed into T. elongatus BP1 and two DNA constructs into Synechocystis 6803. Recent experiments at the molecular level have showed gene expression in GE T. elongatus BP1 carrying the isobutanol production cassette. Outcomes: Three DNA constructs, one designed to produce alcohol, one designed to produce butanol, and one designed for isobutanol production have been verified for successful insertion and incorporation into the cyanobacteria genome. Quantification of isobutanol production by gas chromatography and mass spectrometry is ongoing. Impact: The potential integration of cassettes into the genome of the cyanobacteria indicates an additional and heightened level of bio-risk assessment needed by researchers and the USDA. Task 3. Measure the horizontal gene transfer rate among microorganisms. 100% complete. Progress: In depth studies with GE T. elongatus BP1 were conducted. The condition for growing cyanobacteria and E. coli in co-cultures has been optimized for this study. Studies with these different constructs have completed the goal of examining horizontal gene transfer between GE cyanobacteria T. elongatus BP1 and wild-type E. coli DH5α. Outcomes: Co-culture studies with GE T. elongatus BP1 and, wild-type E. coli were conducted and successful. Antibiotic gene in the form of plasmids within GE T. elongatus BP1 was showed to be transferred into wild-type E. coli within 2 days of co-culturing. The use of the initial growth media has enabled initial studies to go forward and highlighted the need for improved mixtures of growth media conditions. Different mixtures were tested and optimized. Mixture of GE T. elongatus BP1 and E. coli at 1:1 ratio in their respective medium allows the transfer of antibiotic resistant gene in this study. Impact: The results indicate that non-integrative transformants pose a biosafety risk because of the rapid transfer and we aim to determine if integrative transformants undergo less horizontal gene transfer thus potentially directing the development of GE organisms. Objective 2: Complete the assessment on the fate and stability of transgenes 85% accomplished. Task 4. Test the use of a highly thermophilic organism as a biosafety feature to limit the spread of GE organisms into the environment. 100% complete. Progress: The greenhouse on the campus at Old Dominion University was set-up to conduct experiments for this research study. The wild-type T. elongatus BP1 and two GE T. elongatus BP1 were grown in liquid cultures and on solid media. alongside a non-thermophilic cyanobacterial strain in our project. Outcomes: The study showed that 2 weeks of exposure to the cool temperature conditions (15.44-25.30 °C) was enough to cause complete death of GE T. elongatus BP1. However, it took 2-4 weeks for the wild-type T. elongatus BP1 cells to die. Impact: This study revealed that the thermophilic feature of the T. elongatus BP1 may be used to serve as an effective biosafety mechanism at a cool temperature between 15.44°C and 25.30 °C, but may not be able to serve as a biosafety mechanism at warmer temperatures. Task 5. Create a cell-division-controllable photosynthetic organism to limit gene transfer or outcrossing to sexually compatible organisms. 60% complete. This task was redesigned to allow for more extensive assessment of horizontal gene transfer to clinically relevant gram-negative and gram-positive bacteria. The strains studied are E. coli K12, E.coli DH5α, Pseudomonas putida, Staphylococcus epidermis and Bacillus licheniformis. The studies aim to improve our bio-risk assessment of the transformed cyanobacteria BP1-99C. Task 6. Assess the fate and stability of transgenes in designer algae. 95% complete. Progress: A one-year study to assess the stability of GE T. elongatus BP1-pKA (integrative transformation with KIVD-ADH construct) was conducted by monitoring the presence of the transgenes and expression of the kanamycin resistance gene. Outcomes: After one year, the transgenes appear to still be present within GE T. elongatus BP1-pKA cells. PCR of genomic DNA was performed to amplify the insertion site (location on chromosome where foreign genes are inserted), the kanamycin resistance gene. The PCR of the insertion site gave two distinct bands at 2.3 kb and 6.6 kb, indicating the wild-type (or no gene insertion) and insertion of genes, respectively. While the cultures under antibiotic pressure showed the majority of cells still had the transgenes within the insertion site, the cultures without antibiotic pressure showed a gradual increase in cells that did not have the transgenes within the insertion site. Sequencing will be conducted of 1-year sample to assess whether mutations have occurred. Objective 3: Complete the biofuel-producing GE organism survivability tests and demonstrate a biosafety-guarded transgenic alga 90% accomplished. Task 7. Assess the survivability of GE organisms in competition with wild-type cells. 100% complete. Progress: A competition study was conducted by co-culturing a fixed ratio of GE and wild-type cyanobacteria under various environmental conditions and monitoring the change over time. Outcomes: The results indicated that when incubated at room temperature, the GE Synechocystis 6803 does not have a competitive advantage or disadvantage compared to the wild-type Synechocystis 6803. However, in the greenhouse during the cool season (16 oC-26 oC) the wild-type Synechocystis 6803 showed a slight competitive advantage over the GE Synechocystis 6803. Also, under cool season greenhouse conditions, wild-type Synechocystis 6803 was co-cultured with thermophilic GE T. elongatus BP1 BY20 and, wild-type T. elongatus BP1. The GE and wild-type thermophilic strains were outgrown by the Synechocystis 6803 in less than 3 weeks. Task 8. Demonstrate a biosafety-guarded transgenic alga for photosynthetic production of biofuel. 80% complete. Progress: Isobutanol production study was conducted using Thermosynechococcus elongatus BP1. A set of genes were introduced into the cells and isobutanol was detected. Outcomes: The results indicate that the thermophilic strain of cyanobacteria Thermosynechococcus elongatus BP1 is able to produce biofuel (isobutanol). In combination with results from task 4, it is expected that biofuel production using thermophilic cyanobacteria provides a built-in biosafety feature. Theses genetically modified cells would not likely to survive if they escaped containment of high temperature which would significantly reduce the risk of gene transfer.

Publications

Type: Journal Articles Status: Published Year Published: 2019 Citation: Nguyen TH, Barnes CL, Agola JP, Sherazi S, Greene LH, Lee JW (2019) Demonstration of horizontal gene transfer from genetically engineered Thermosynechococcus elongatus BP1 to wild-type E. coli DH5, Gene, 704: 49-58.
Type: Journal Articles Status: Published Year Published: 2019 Citation: Lee JW (2019) Protocol measuring horizontal gene transfer from algae to non-photosynthetic organisms, MethodsX 6:15641574.
Type: Journal Articles Status: Published Year Published: 2020 Citation: Sacko O, Barnes CL, Greene LH, Lee JW (2020) Survivability of Wild-Type and Genetically Engineered Thermosynechococcus elongatus BP1 with Different Temperature Conditions, Applied Biosafety: Journal of ABSA International, 25(2): 104-117.

Progress 09/01/18 to 08/31/19

Outputs
Target Audience:The scientific community, policy makers and the photosynthetic biofuel and bioproduct industry. Changes/Problems:This project has recently been extended into year 4 based on the USDA-approved non-cost extension of this project to August 31, 2020. What opportunities for training and professional development has the project provided?In Task 1 three graduate students gained advanced scientific training in genomic analysis. In Task 2 three graduate students gained advanced scientific training in molecular biology and genetic engineering techniques. In Task 3 three graduate students have gained advanced scientific training in microbiology and molecular biology techniques. Since initiating Task 3, two undergraduates (one biology major and one biochemistry post-baccalaureate) have joined the team and are gaining scientific training in this field. In Task 4, three additional undergraduate students (two biology majors and one chemistry major) gained training in microbiology. The research team is comprised of the following students: Thu Nguyen, Graduate Student; Cherrelle Barnes, Graduate Student; Oumar Sacko, Graduate Student; Megan Hept, Graduate Student; Andrea Zourou, Graduate Student; Simirjeet Singh, Graduate Student. Two graduate students, one undergraduate and the post-baccalaureate research are co-authors on a manuscript currently published. Two graduate students are currently co-authors on a manuscript currently under review. How have the results been disseminated to communities of interest?Some of the results have now been presented in the national conferences and published in the peer-reviewed international journals. More results are being prepared for submission to a national conference and for publication in international journals. What do you plan to do during the next reporting period to accomplish the goals?In consistence with the project timeline as shown in Table 1 of the original project proposal and with the recently approved non-cost extension of the project to year 4 until August 31, 2020, we aim to be 100% completed with all three objectives by year 4. Tasks 3 and 4 are now 100% complete. Tasks 1-2 and 6 will be completed within the first five months of year 4. We will advance and complete Task 5 (Create a cell-division-controllable photosynthetic organism to limit gene transfer or outcrossing to sexually compatible organisms) throughout the year 4. It involves designing new constructs with DNAstar Lasergene software and CLC Bio and in consideration of biochemical pathways, gene regulation and membrane protein biochemistry/structural biology/computational biochemistry. We will continue Task 7 (Assess the survivability of GE organisms with its wild-type cells) through the co-culturing studies in Year 4. Biofuel production will also continue to be assessed using techniques such as GC x GC/MS. The constructs designed in Year 2 will be upgraded as required to accomplish this aim. Task 8 (Demonstrate a biosafety-guarded transgenic alga for photosynthetic production of biofuel) will be completed in Year 4. Furthermore, we will present our results at both scientific conferences and through submission to top journals for publication. We aim to publish the results of the fate and stability studies. In 2020 results will be presented at a National Conference. This project currently supports the thesis research work of six graduate students (Thu Nguyen, Cherrelle Barnes, Oumar Sacko, Megan Hept, Andrea Zourou and Simirjeet Singh). The PI (James Lee) and the Co-PI (Lesley Greene) aim to also present research findings at a scientific conference and develop a graduate-level course.

Impacts
What was accomplished under these goals? Objective 1: Create special transgenic organisms to assess survivability and horizontal gene transfer (Year 1) 95% accomplished. Task 1. Synthesize DNA constructs to generate genetically engineered (GE) microorganisms for measuring horizontal gene transfer. (2017-2019) 95% complete. Progress: Seven genetic constructs called DNA cassettes were designed to contain genes and regulatory signals for production of biofuels and other products to monitor the behavior of the genetic material inside the cyanobacteria. This year, the eighth DNA construct was designed and synthesized for production of isobutanol which is a high potential biofuel source. Outcomes: Seven DNA cassettes were synthesized. Each cassette additionally contains signals such as promoters, termination signals to enable the expression of the foreign genes as well as restriction sites and recombination sites for integration into the genome of the target organisms. The eighth DNA cassette was designed utilizing a commonly used promoter (IPTG) in synthetic biology along with other mentioned components. Impact: The methodology developed forms the foundation for the remaining seven tasks in this research study and establishes an approach for rationale design of GE cyanobacteria for biofuel production, risk assessment and containment. Task 2. Create GE organisms by genetic transformation. (2017-2019) 90% complete. Progress: Five genetic constructs were successfully transformed into T. elongatus BP1 and two DNA constructs into Synechocystis 6803. Experimental conditions have been developed to identify the presence of the GE molecular genetic construct inside the organism as a circular piece of DNA called a plasmid (non-integrative transformant) and as an integrated cassette in the cyanobacterial genome (integrated transformant) which means the foreign DNA we designed was inserted into the cyanobacterial genome. Recent experiments at the molecular level have showed gene expression in GE T. elongatus BP1 carrying the isobutanol production cassette. Outcomes: Three DNA constructs, one designed to produce alcohol, one designed to produce butanol, and one designed for isobutanol production (KIVD-ADH, KIVD-BDH, and pB-BP1KY respectively) have been verified for successful insertion and incorporation into the cyanobacteria genome. Quantification of isobutanol production from pB-BP1KY by gas chromatography and mass spectrometry is ongoing. The two new constructs that were synthesized and successfully transformed in Synechocystis sp. strain PCC 6803 contained in one, a methanol-producing gene (MeOH-Strep) and in the other a yellow fluorescence protein as well as a lipase producing gene (YFP-R-Lipase). Impact: The potential integration of cassettes into the genome of the cyanobacteria indicates an additional and heightened level of bio-risk assessment needed by researchers and the USDA. Task 3. Measure the horizontal gene transfer rate among microorganisms. (2017-2019) 100% complete. Progress: In depth studies with GE T. elongatus BP1 containing the KIVD-BDH and KIVD-ADH constructs were conducted. The condition for growing cyanobacteria and E. coli in co-cultures has been optimized for this study. Studies with these different constructs have completed the goal of examining horizontal gene transfer between GE cyanobacteria T. elongatus BP1 and wild-type E. coli DH5α. Outcomes: Co-culture studies with GE T. elongatus BP1 containing the KIVD-BDH and KIVD-ADH and wild-type E. coli were conducted and successful. Antibiotic gene in the form of plasmids within GE T. elongatus BP1 was showed to be transferred into wild-type E. coli within 2 days of co-culturing. The use of the initial growth media has enabled initial studies to go forward and highlighted the need for improved mixtures of growth media conditions. Different mixtures were tested and optimized. Mixture of GE T. elongatus BP1 and E. coli at 1:1 ratio in their respective medium allows the transfer of antibiotic resistant gene in this study. Impact: The results indicate that non-integrative transformants (those with free DNA constructs inside the cyanobacteria) pose a biosafety risk because of the rapid transfer and we aim to determine if integrative transformants (those with the DNA constructs inserted into the host genome) undergo less horizontal gene transfer thus potentially directing the development of GE organisms. Objective 2: Complete the assessment on the fate and stability of transgenes (Year 2) 60% accomplished. Task 4. Test the use of a highly thermophilic organism as a biosafety feature to limit the spread of GE organisms into the environment. (2017-2019) 100% complete. Progress: The greenhouse on the campus at Old Dominion University was set-up to conduct experiments for this research study. The wild-type T. elongatus BP1 and two GE T. elongatus BP1 were grown in liquid cultures and on solid media. alongside a non-thermophilic cyanobacterial strain (Synechocystis 6803) in our project. Outcomes: The study showed that 2 weeks of exposure to the cool temperature conditions (15.44-25.30 °C) was enough to cause complete death of GE T. elongatus BP1. However, it took 2-4 weeks for the wild-type T. elongatus BP1 cells to die. Impact: This study revealed that the thermophilic feature of the T. elongatus BP1 may be used to serve as an effective biosafety mechanism at a cool temperature between 15.44°C and 25.30 °C, but may not be able to serve as a biosafety mechanism at warmer temperatures. Task 5. Create a cell-division-controllable photosynthetic organism to limit gene transfer or outcrossing to sexually compatible organisms. (2017-2019) 10% complete. Progress: Gene design is underway. The main effort of this work will be undertaken in year 4 work activities. Task 6. Assess the fate and stability of transgenes in designer algae. (2017-2019) 80% complete. Progress: A one-year study to assess the stability of GE T. elongatus BP1-pKA (integrative transformation with KIVD-ADH construct) was conducted by monitoring the presence of the transgenes and expression of the kanamycin resistance gene. Outcomes: After one year, the transgenes appear to still be present within GE T. elongatus BP1-pKA cells. PCR of genomic DNA was performed to amplify the insertion site (location on chromosome where foreign genes are inserted), the kanamycin and resistance gene. The PCR of the insertion site gave two distinct bands at 2.3 kb and 6.6 kb, indicating the wild-type (or no gene insertion) and insertion of genes, respectively. While the cultures under antibiotic pressure showed the majority of cells still had the transgenes within the insertion site, the cultures without antibiotic pressure showed a gradual increase in cells that did not have the transgenes within the insertion site. Objective 3: Complete the biofuel-producing GE organism survivability tests and demonstrate a biosafety-guarded transgenic alga (Year 3) 50% accomplished. Task 7. Assess the survivability of GE organisms in competition with wild-type cells. (2017-2019) 60% complete. Progress: A competition study was conducted by co-culturing GE and wild-type cyanobacteria in a fixed ratio and monitoring the change in ratio over time. Outcomes: The results indicated that when incubated at room temperature, the wild-type Synechocystis 6803 and the GE Synechocystis 6803 (6803-YFP-R-Lipase) grew at the same rate; they each maintained their initial ratio (2:3) to the wild-type Synechococcus elongatus 7942 from day 1 to day 28 of incubation. However, in the greenhouse during the cool season the wild-type Synechocystis 6803 showed a slight competitive advantage over the GE Synechocystis 6803 (6803-YFP-R-Lipase). Task 8. Demonstrate a biosafety-guarded transgenic alga for photosynthetic production of biofuel. (2017-2019) 0% complete. There is nothing yet to report here at this time. This Task will be undertaken in year 4 work activities.

Publications

Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Published Abstracts Citations: 1) Bio-risk assessment research on genetically engineered cyanobacteria for sustainable biofuel (2019) Nguyen TH, Barnes CL, Greene LH, Lee JW (2019), Experimental Biology 2019, Orlando, FL Abstract ID: lb301 2) Competition study between wild type cyanobacteria Synechococcus elongatus PCC 7942 and plasmid transformant Synechocystis PCC 6803 (2019) Sacko O, Greene LH, Lee JW, American Chemical Society National Meeting (Biochemical Technology) 2019, Orlando, FL Abstract ID: 3126326 3) Survivability of genetically engineered Thermosynechococcus elongatus BP1 in different temperature conditions (2019) Sacko O, Barnes CL, Greene LH, Lee JW, American Chemical Society National Meeting (Environmental Chemistry) 2019, Orlando, FL Abstract ID: 3128391 4) Assessing the Stability and Expression of Transgenes in Genetically Engineered Cyanobacteria for Biofuel Production (2019) Barnes CL, Lee JW, Greene LH, American Chemical Society National Meeting (Biochemical Technology) 2019, Orlando, FL Abstract ID: 3128212 5) Designer biosynthetic pathways for photosynthetic biofuels and bioproducts: opportunities and challenges, Lee JW, poster presented in the Experimental Biology Conference (April 6-9, 2019) sponsored by American Society for Biochemistry and Molecular Biology in Orlando, FL., Abstract Number:486.1 Published Online:1 April 2019 in The FASEB Journal, https://www.fasebj.org/doi/abs/10.1096/fasebj.2019.33.1_supplement.486.1 Abstract ID: 486.1
Type: Journal Articles Status: Published Year Published: 2019 Citation: Published manuscripts: 1) Nguyen TH, Barnes CL, Agola JP, Sherazi S, Greene LH, Lee JW (2019) Demonstration of horizontal gene transfer from genetically engineered Thermosynechococcus elongatus BP1 to wild-type E. coli DH5, Gene, 704: 49-58. 2) Lee JW (2019) Protocol measuring horizontal gene transfer from algae to non-photosynthetic organisms, MethodsX 6:15641574.
Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Submitted manuscript under review: 1) Oumar Sacko, Cherrelle L. Barnes, Lesley H. Greene and James W. Lee (2019) Survivability of Wild Type and Genetically Engineered Thermosynechococcus elongatus BP1 with Different Temperature Conditions in Winter and Spring Seasons, submitted for peer-reviewed journal publication.

Progress 09/01/17 to 08/31/18

Outputs
Target Audience:The scientific community, policy makers and the photosynthetic biofuel and bioproduct industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In Task 1 three graduate students gained advanced scientific training in genomic analysis. In Task 2 three graduate students gained advanced scientific training in molecular biology and genetic engineering techniques. In Task 3 three graduate students have gained advanced scientific training in microbiology and molecular biology techniques. Since initiating Task 3, two undergraduates (one biology major and one biochemistry post-baccalaureate) have joined the team and are gaining scientific training in this field. In Task 4, three additional undergraduate students (two biology majors and one chemistry major) gained training in microbiology. The research team is comprised of the following students: Thu Nguyen, Graduate Student; Cherrelle Barnes, Graduate Student; Oumar Sacko, Graduate Student; Sana Sherazi, Undergraduate Student; An Ha, Undergraduate Student; Kevin Nguyen, Undergraduate Student; Bryan Koury, Undergraduate Student; Jason Agola, Post-baccalaureate researcher. Two graduate students, one undergraduate and the post-baccalaureate research are co-authors on a manuscript currently under review. How have the results been disseminated to communities of interest?The results are being prepared for submission to a national conference and for publication in international journals. What do you plan to do during the next reporting period to accomplish the goals?In consistence with the project timeline as shown in Table 1 of the original project proposal, by year 3, we aim to be 100% completed with all three objectives. Further, we will present our results at both scientific conferences and through submission to top journals for publication. Tasks 1-4 and 6 will be completed within the first three months of year 3. We will advance and complete Task 5 (Create a cell-division-controllable photosynthetic organism to limit gene transfer or outcrossing to sexually compatible organisms) throughout the year. It involves designing new constructs with CLC Bio and in consideration of biochemical pathways, gene regulation and membrane protein biochemistry/structural biology/computational biochemistry. We will continue Task 7 (Assess the survivability of GE organisms with its wild-type cells) through the co-culturing studies in Year 3. Biofuel production will also continue to be assessed using techniques such as GC x GC/MS. The constructs designed in Year 2 will be upgraded as required to accomplish this aim. Task 8 (Demonstrate a biosafety-guarded transgenic alga for photosynthetic production of biofuel) will be completed in Year 3. We aim to publish the results of the horizontal gene transfer studies and the manuscript is under review. In March 2019 three graduate students (Thu Nguyen, Cherrelle Barnes and Oumar Sacko) will attend the American Chemical Society Conference (Chemistry for New Frontiers) in Orlando, Florida and present the results of the growth study, survivability/competition studies and horizontal gene transfer studies. Three undergraduate students will be trained and will present at the Virginia Academy of Sciences Annual Conference in May 2019. The PI (James Lee) and the Co-PI (Lesley Greene) aim to also present research findings at a scientific conference and develop a graduate-level course.

Impacts
What was accomplished under these goals? Objective 1: Create special transgenic organisms to assess survivability and horizontal gene transfer (Year 1) 95% accomplished. Task 1. Synthesize DNA constructs to generate genetically engineered (GE) microorganisms for measuring horizontal gene transfer. (2017-2019) 90% complete. Progress: Computational studies were conducted to extensively analyze the genomes of the three model cyanobacteria (Synechocystis sp. strain PCC 6803, Synechococcus elongatus PCC 7942, Thermosynechococcus elongatus BP-1) using advanced genomics software. Seven genetic constructs called DNA cassettes were designed to contain genes and regulatory signals for the production of biofuels and other products to monitor the behavior of the genetic material inside the cyanobacteria. Based on the studies conducted this past year an eighth construct is currently being designed. Outcomes: The computational studies provided insight into which locations could support the insertion of the foreign genes. Seven DNA cassettes were synthesized. Two were from preliminary work and five in the present research project. They were created in a modular design so that different coding elements (antibiotic resistance, alcohol producing genes, fluorescent proteins and fluorescent tags) could be added or removed. Each cassette additionally contains signals such as promoters, termination signals to enable the expression of the foreign genes as well as restriction sites and recombination sites for integration into the target organisms. An eighth cassette is currently being designed to facilitate expression of the product. Impact: The methodology developed forms the foundation for the remaining seven tasks in this research study and establishes an approach for rationale design of GE cyanobacteria for biofuel production, risk assessment and containment. Task 2. Create GE organisms by genetic transformation. (2017-2019) 80% complete. Progress: Four genetic constructs were successfully transformed into T. elongatus BP1 and two genetic construct into Synechocystis 6803. Upon completion of the construction of the remaining three clones from Task 1, the final transformation experiments will be conducted. Experimental conditions have been developed to identify the presence of the GE construct inside the organism as a circular piece of DNA called a plasmid (non-integrative transformant) and as an integrated cassette in the cyanobacterial genome (integrated transformant) which means the foreign DNA we designed was inserted into the cyanobacterial genome. Outcomes: Conditions for the insertion of the genetic constructs and a method for detecting if the insertion was inside the cyanobacteria only or further incorporated into the genome by a process termed homologous recombination was established and further refined. Two DNA constructs, one which is designed to produce alcohol and the other designed to produce butanol (KIVD-ADH and KIVD-BDH, respectively) have been verified for successful insertion and incorporation into the cyanobacteria genome. A manuscript presenting these results is under review. The two new constructs that were synthesized and successfully transformed contained in one, a methanol-producing gene (MeOH-Strep) and in the other a yellow fluorescence protein as well as a lipase producing gene (YFP-R-Lipase). Impact: The methodology developed forms the foundation for the remaining six tasks in this research project and establishes an approach for rationale design of GE cyanobacteria for biofuel production, risk assessment and containment. The potential integration of cassettes into the genome of the cyanobacteria indicates an additional and heightened level of bio-risk assessment needed by researchers and the USDA. Task 3. Measure the horizontal gene transfer rate among microorganisms. (2017-2019) 80% complete. Progress: In depth studies with GE T. elongatus BP1 containing the KIVD-BDH and KIVD-ADH constructs were conducted. The conditions for growing cyanobacteria and E. coli in co-cultures has been optimized for this study. Outcomes: Co-culture studies with GE T. elongatus BP1 containing the KIVD-BDH and KIVD-ADH and wild-type E. coli were conducted and successful. The use of the initial growth media has enabled initial studies to go forward and highlighted the need for improved mixtures of growth media conditions. Different mixtures were tested and optimized. Impact: The results indicate that non-integrative transformants (those with free DNA constructs inside the cyanobacteria) pose a biosafety risk because of the rapid transfer and we aim to determine if integrative transformants (those with the DNA constructs inserted into the host genome) undergo less horizontal gene transfer thus potentially directing the development of GE organisms. A manuscript presenting these results is under review. Objective 2: Complete the assessment on the fate and stability of transgenes (Year 2) 40% accomplished. Task 4. Test the use of a highly thermophilic organism as a biosafety feature to limit the spread of GE organisms into the environment. (2017-2019) 95% complete. Progress: The greenhouse on the campus at Old Dominion University was initially set-up to conduct experiments for this research study. T. elongatus BP1 is currently being grown in liquid cultures and on solid media alongside the two non- thermophilic cyanobacterial strains in our project. Outcomes: The results indicate that Synechocystis 6803 are growing while T. elongatus BP1 growth was dependent on the time of the year. The growth study was conducted in the greenhouse on the BP1 BY20 (Integrative transformant with YFP-R-Lipase) during different times interval from February to May. BP1 wild-type and the BP1 containing integrated YFP-R-Lipase were not able to grow in the greenhouse after 4 weeks. BP1 survivability assay study was also conducted to assess the survival of the BP1 cells incubated in the greenhouse by inoculating them in the Percival after one week intervals of growth (up to four weeks) in the greenhouse. Impact: The results indicate that temperature can be a limiting factor to the growth of GE cyanobacteria in the environment for BP1 should it escape containment in hot versus cooler environmental temperatures. Task 5. Create a cell-division-controllable photosynthetic organism to limit gene transfer or outcrossing to sexually compatible organisms. (2017-2019) 10% complete. Progress: Gene design is underway. The main effort of this work will be undertaken in year 3 work activities. Task 6. Assess the fate and stability of transgenes in designer algae. (2017-2019) 15% complete. Progress: Preliminary studies have been conducted to assess the expression of fluorescence labeled protein in the engineered cyanobacteria by fluorescence microscopy. Outcomes: Due to the auto-fluorescence no distinction can be made between the WT organism and the expressed protein at the present. We are working to optimize the system and methodology to detect the expressed fluorescent tagged protein. Objective 3: Complete the biofuel-producing GE organism survivability tests and demonstrate a biosafety-guarded transgenic alga (Year 3) 5% accomplished. Task 7. Assess the survivability of GE organisms in competition with wild-type cells. (2017-2019) 10% complete. Progress: This work is currently being conducted. We expect to have preliminary results in mid-September and refine the studies based on the data to complete this task. Task 8. Demonstrate a biosafety-guarded transgenic alga for photosynthetic production of biofuel. (2017-2019) 0% complete. There is nothing yet to report here at this time. This Task will be undertaken in year 3 work activities.

Publications

Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Published Abstract: Demonstration of Horizontal Gene Transfer from Genetically Engineered Cyanobacteria to Wild Type E. Coli (2018) T. Nguyen, C. Barnes, J. Agola, S. Sherazi, L.H. Greene, J.W. Lee. 255th American Chemical Society National Meeting, New Orleans, LA. Paper ID #2864673

Progress 09/01/16 to 08/31/17

Outputs
Target Audience:The scientific community, policy makers and the photosynthetic biofuel and bioproduct industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In Task 1 three graduate students gained advanced scientific training in genomic analysis. In Task 2 three graduate students gained advanced scientific training in molecular biology and genetic engineering techniques. In Task 3 three graduate students have gained advanced scientific training in microbiology and molecular biology techniques. Since initiating Task 3, two undergraduates (one biology major and one biochemistry post-baccalaureate) have joined the team and are gaining scientific training in this field. The research team is comprised of the following students: Thu Nguyen, Graduate Student; Cherrelle Barnes, Graduate Student; Oumar Sacko, Graduate Student; Sana Sherazi, Undergraduate Student; Jason Agola, Post-baccalaureate researcher. How have the results been disseminated to communities of interest?The results are being prepared for submission to a national conference and for publication in an international journal. What do you plan to do during the next reporting period to accomplish the goals?In consistence with the project timeline as shown in Table 1 of the original project proposal, by year 2, we aim to be ~95% completed with Task 1 and significantly advance Tasks 2-4. In Year 2 the use of photobioreactors will also be implemented in the green house to accomplish Task 4. We will also establish the foundation for Task 5 (Create a cell-division-controllable photosynthetic organism to limit gene transfer or outcrossing to sexually compatible organisms) and involves designing new constructs with CLC Bio and in consideration of biochemical pathways, gene regulation and membrane protein biochemistry/structural biology/computational biochemistry. Task 6 (Assess the fate and stability of transgenes in designer algae) will be conducted in parallel with Task 4. Objective 3 (Complete the biofuel-producing GE organism survivability tests that demonstrate a biosafety-guarded transgenic alga) will be accomplished in Year 3. We will begin Task 7 (Assess the survivability of GE organisms with its wild-type cells) through the co-culturing studies in Year 2. Biofuel production will also continue to be assessed using techniques such as GC x GC/MS. The constructs designed in Year 1 will be upgraded as required to accomplish this aim. Task 8 (Demonstrate a biosafety-guarded transgenic alga for photosynthetic production of biofuel) will be completed in Year 3. Tasks 4 and 5 lay the foundation for the completion of Task 8. We aim to publish the results of the horizontal gene transfer studies. In March 2018 three graduate students (Thu Nguyen, Cherrelle Barnes and Oumar Sacko) will attend the American Chemical Society Conference entitled 'Nexus of Food, Energy & Water' in New Orleans, LA and present the results of the construction of the GE organisms and horizontal gene transfer studies. The abstract to be submitted to the Biological Chemistry section will also contains the names of the two undergraduate students (Sana Sherazi and Jason Agola) who participated in these studies. Three additional undergraduate students will also be trained. They are Kevin Nguyen (undergraduate), Bryan Koury (undergraduate) and An Ha (Undergraduate). The PI (James Lee) and the Co-PI (Lesley Greene) aim to also present research findings at a scientific conference and develop a graduate-level course.

Impacts
What was accomplished under these goals? Objective 1: Create special transgenic organisms to assess survivability and horizontal gene transfer (Year 1) 90% accomplished. Task 1. Synthesize DNA constructs to generate genetically engineered (GE) microorganisms for measuring horizontal gene transfer. (2017-2019) 60% complete. Progress: Computational studies were conducted to extensively analyze the genomes of the three model cyanobacteria (Synechocystis sp. strain PCC 6803, Synechococcus elongatus PCC 7942, Thermosynechococcus elongatus BP-1) using advanced genomics software. Seven genetic constructs called DNA cassettes were designed to contain genes and regulatory signals for the production of biofuels and other products to monitor the behavior of the genetic material inside the cyanobacteria. Outcomes: The computational studies provided insight into which locations could support the insertion of the foreign genes. Seven DNA cassettes were synthesized. Two were from preliminary work and five in the present research project. They were created in a modular design so that different coding elements (antibiotic resistance, alcohol producing genes, fluorescent proteins and fluorescent tags) could be added or removed. Each cassette additionally contains signals such as promoters, termination signals to enable the expression of the foreign genes as well as restriction sites and recombination sites for integration into the target organisms. Additional DNA constructs will be designed and synthesized based on the results of future tasks. Impact: The methodology developed forms the foundation for the remaining seven tasks in this research study and establishes an approach for rationale design of GE cyanobacteria for biofuel production, risk assessment and containment. Task 2. Create GE organisms by genetic transformation. (2017-2019) 30% complete. Progress: Three genetic constructs were successfully transformed into T. elongatus BP1 and one genetic construct into Synechocystis 6803. Upon completion of the construction of the remaining three clones from Task 1, the final transformation experiments will be conducted. Experimental conditions have been developed to identify the presence of the GE construct inside the organism as a circular piece of DNA called a plasmid (non-integrative transformant) and as an integrated cassette in the cyanobacterial genome (integrated transformant) which means the foreign DNA we designed was inserted into the cyanobacterial genome. Outcomes: Conditions for the insertion of the genetic constructs and a method for detecting if the insertion was inside the cyanobacteria only or further incorporated into the genome by a process termed homologous recombination was established. Two DNA constructs, one which is designed to produce alcohol and the other designed to produce butanol (KIVD-ADH and KIVD-BDH, respectively) have been verified for successful insertion and incorporation into the cyanobacteria genome. Impact: The methodology developed forms the foundation for the remaining six tasks in this research project and establishes an approach for rationale design of GE cyanobacteria for biofuel production, risk assessment and containment. Task 3. Measure the horizontal gene transfer rate among microorganisms. (2017-2019) 20% complete. Progress: Initial studies with GE T. elongatus BP1 containing the KIVD-BDH and KIVD-ADH constructs were conducted. Optimizing the conditions for growing cyanobacteria and E. coli in co-cultures is underway. Outcomes: Co-culture studies with GE T. elongatus BP1 containing the KIVD-BDH and KIVD-ADH and wild-type E. coli were initiated and successful. The use of the initial growth media has enabled initial studies to go forward and highlighted the need for improved mixtures of growth media conditions. Investigations are underway to test different mixtures. Following optimization of the media we anticipate success for the other GE constructs. Impact: Preliminary results indicate at present non-integrative transformants (those with free DNA constructs inside the cyanobacteria) pose a biosafety risk because of the rapid transfer and we aim to determine if integrative transformants (those with the DNA constructs inserted into the host genome) undergo less horizontal gene transfer thus potentially directing the development of GE organisms. Objective 2: Complete the assessment on the fate and stability of transgenes (Year 2) 2% accomplished. Task 4. Test the use of a highly thermophilic organism as a biosafety feature to limit the spread of GE organisms into the environment. (2017-2019) 5% complete. Progress: The greenhouse on the campus at Old Dominion University was initially set-up to conduct experiments for this research study. T. elongatus BP1 is currently being grown in liquid cultures and on solid media alongside the two non-thermophilic cyanobacterial strains in our project. Outcomes: The preliminary initial results indicate that Synechocystis 6803 and S. elongatus 7942 are growing while T. elongatus BP1 has not shown significant growth as expected. Task 5. Create a cell-division-controllable photosynthetic organism to limit gene transfer or outcrossing to sexually compatible organisms. (2017-2019) 0% complete. There is nothing yet to report here at this time, since this Task is year 2-3 work activities. Task 6. Assess the fate and stability of transgenes in designer algae. (2017-2019) 0% complete. There is nothing yet to report here at this time, since this Task is year 2-3 work activities. Objective 3: Complete the biofuel-producing GE organism survivability tests and demonstrate a biosafety-guarded transgenic alga (Year 3) 0% accomplished. Task 7. Assess the survivability of GE organisms in competition with wild-type cells. (2017-2019) 0% complete. There is nothing yet to report here at this time, since this Task is year 2-3 work activities. Task 8. Demonstrate a biosafety-guarded transgenic alga for photosynthetic production of biofuel. (2017-2019) 0% complete. There is nothing yet to report here at this time, since this Task is year 2-3 work activities.

Publications

Type: Conference Papers and Presentations Status: Under Review Year Published: 2017 Citation: Manuscripts will be submitted to journals for publication in Year 2. Abstracts will be submitted for presentation in conferences in Year 2.