Dr. Miller REM Projects (UMBC)

Biofuels are obtained from biomass and are a source of renewable energy that is more sustainable than fuels derived from petroleum. We aim to make algal based biofuels more efficient by increasing the growth rate and lipid production in algae. Algae are limited for uptake of carbon dioxide (CO2), which has limited solubility in water. We hypothesize that certain enzymes that concentrate CO2 in algae are limiting for growth, and we predict that overexpressing them will speed growth and biomass production. Chlamydomonas reinhardtii, a green microalga, is a model organism that can be genetically manipulated to overexpress certain proteins. This investigation focuses on CAH3, a carbonic anhydrase that catalyzes the conversion of carbonic acid into CO2, increasing its concentration in the chloroplast pyrenoid, where it is fixed into sugars by the enzyme rubisco. We first designed and ordered a synthetic CAH3 coding region (with HA epitope tag sequence at 3’ end) flanked by XhoI and BamHI restriction sites at its 5’ and 3’ ends. Respectively. Then we used restriction digests to isolate this gene fragment, and we used DNA ligase to clone it into our C. reinhardtii expression vector (pUC-ARG) to produce plasmid pUC-ARG-CAH3. After transforming this construct into C. reinhardtii strain CC4350, we used western blot analysis with anti-HA antibody to test for CAH3 abundance, but none of the transformants we tested had detectable levels of CAH3 protein. In an attempt to improve expression, we next replaced the ARG7 selectable marker gene with a bleomycin resistance cassette (ble-2A) that couples the expression of the ble protein (through a viral 2a-peptide sequence) to the CAH3 sequence. To this end we digested pUC-ARG-CAH3 with EcoRV to remove the ARG7 gene, religated, then digested with XhoI and ligated a gene-synthesized ble-2A fragment flanked with XhoI sites into the expression vector. Restriction analysis and sequencing of plasmid minipreps confirmed that one ligation product had the fragment cloned in correct orientation. We are now using electroporation to transform this plasmid into C. reinhardtii, then we will use western blot analysis to test transformants for protein expression. If transformants are obtained that overexpress CAH3, we will use an algal multicultivator to compare their growth rate to the wild type using the muti-cultivator. Consequently, if increased growth is achieved, the same procedure can be applied to the industrial alga Chlorella vulgaris for production of biofuels.

Biofuels are derived from organic matter and are hence renewable energy sources. There are several kinds of biofuels, but algal-based biofuels are particularly promising because algae use carbon dioxide (CO2) as carbon source, can grow on wastewater, and don’t require farmable land. Algal biofuels are not yet competitive with fossil fuels in part because algae do not grow rapidly under normal growth conditions. CO2 is poorly soluble in water and hence limiting for algal growth, and we hypothesize that increasing expression of proteins that improve uptake of CO2 will speed growth and biomass production. We are testing this hypothesis by overexpressing the LCI1 protein in Chlamydomonas reinhardtii, a single celled green microalga that is easily genetically manipulated, to overexpress. LCI1 is a plasma membrane transporter that brings carbonate into the cell; carbonate is later converted into CO2 by carbonic anhydrases. To overexpress LCI1, we used XhoI and BamHI restriction sites to clone a synthetic LCI1 coding fragment (with MYC epitope tag at the 3’end of the gene) into a C. reinhardtii expression vector to generate pUC-ARG-LCI1. We transformed this plasmid into C. reinhardtii and used western blot analysis with anti-MYC antibody to test for expression of LCI1, but we could not detect transgenic protein in any of the transformants. In an attempt to improve expression, we next used an EcoRV digestion to remove the ARG7 selectable marker gene from pUC-ARG-LCI1 and ligated a ble-2A DNA fragment with XhoI sites at its termini into the XhoI site just downstream the start codon in the LCI1 coding sequence. The ble-2A fragment encodes a protein that provides resistance to the antibiotic bleomycin and couples expression of that protein to that of the downstream protein, in this case LCI1. We tested 18 ligation products by restriction analysis and sequencing and found one with the ble-2A fragment in the proper orientation. Next we will transform this new plasmid into C. reinhardtii, and we will use western blot analysis to test for protein expression. The transformants that show overexpression will be tested for growth rate and biomass production compared to the wild type. If increased growth/biomass is achieved, the same procedure could be used for Chlorella vulgaris, a green alga better suited for industrial production of biofuels.

As reliance on fossil fuels has become increasingly detrimental to our environment, renewable energy sources such as biofuels have emerged as legitimate alternatives. One of the most promising replacements for petroleum is biodiesel derived from algae. Algal biofuels are energy dense, their production does not compete with food crops, and the primary resources needed to produce them (CO2 and light) are free, but currently algal biofuels are too expensive to be commercially viable, in part because the algae they are produced from do not grow fast enough. We hypothesize that a limiting factor for growth rate is uptake of CO2 from water, and that overexpressing membrane carbonate transporters will increase growth, because carbonate is later converted to CO2. We are using the model organism Chlamydomonas reinhardtii, a green microalga that is tractable at the molecular genetic level and has a fully sequenced genome, to test this idea. This study focuses on NAR1.2, a transporter that moves carbonate from the cytoplasm into the chloroplast. We gene synthesized the NAR1.2 coding region (with FLAG epitope tag sequence at 3’ end) to contain XhoI and BamHI restriction sites at its termini, then used these enzymes to isolate this gene fragment and clone it into our C. reinhardtii expression vector (pUC-ARG) digested with the same enzymes, to produce plasmid pUC-ARG-NAR1.2. We transformed this construct into recipient C. reinhardtii strain CC4350, and used western blot analysis with anti-FLAG antibody to analyze transgenic protein abundance, but none of the transformants accumulated NAR1.2 protein. Now we are modifying this vector by inserting a ble-2a peptide fragment upstream of the NAR 1.2 coding region to couple expression of the bleomycin resistance protein to expression of NAR1.2, a strategy that has worked in our lab for other proteins. First we digested pUC-ARG-NAR1.2 with restriction enzyme EcoRV to remove the 7.6-kb ARG7 selectable marker cassette, generating pUC-NAR1.2-noARG. Now we are subcloning an ~600-bp XhoI restriction fragment with ble-2a sequence just upstream the start codon of NAR1.2 in this plasmid. Next we will electroporate the new NAR 1.2 construct into C. reinhardtii, use western blot analysis to test for the expression of NAR 1.2 protein, and test the growth rate compared to wild type C. reinhardtii. If we are successful, ultimately we will apply this methodology to increase the growth of industrial species such as Chlorella vulgaris, to improve the economic viability of algal biofuels.

Biofuels are renewable, environmentally friendly, and produced from organic materials rather than fossil fuels. Algal biofuels have advantages over plant-based biofuels for a number of reasons: algae grow faster than land plants, algae do not require pure water, and algae do not require fertile land. Additionally, algae have many genetic manipulation capabilities. Chlamydomonas reinhardtii, a unicellular eukaryotic green alga, is the most widely used research model for algal biofuels, with an entirely sequenced genome that is largely annotated. The gene used for this research, DGTT1, encodes a DGAT enzyme responsible for converting diacylglycerol (DAG) to triacylglycerol (TAG).

Biofuels are fuels that are sustainably produced from biological material and can be used to replace fossil fuels, which release pollutants and are in limited supply. Producing biofuels from algae will lessen fossil fuel demand, and the impact of greenhouse emissions. Algae are renewable, and can produce lipids that can be used to produce biofuels. Algae can be cultivated on wastewater not suitable for human consumption while absorbing CO2 from the atmosphere. Chlamydomonas reinhardtii is a unicellular photosynthetic green algae used for research in production of algal biofuels. Its fully mapped and largely annotated sequence, and relatively rapid growth makes this organism the best available model for biofuel research. This study focuses on the Carbonic Anhydrase 1 (CAH1) enzyme that converts CO2 into HCO3- in the cell periplasm. Our prediction is that over expression of this gene will increase the algal biomass by improving CO2 uptake. Using recombinant DNA technology, we ligated a synthesized CAH1 gene fragment into a C. reinhardtii expression vector and transformed into C. reinhardtii. None of the transformants expressed CAH1 protein so we modified the vector by ligating a ble-2A peptide fragment that permits easy selection for overexpressing transformants. One ligation product was verified by sequencing to have the ble-2A peptide fragment ligated in the correct orientation, and we are electroplating into C. reinhardtii. We will do western-blot analysis on transformants to determine transgenic protein expression. We will then use an algal multicultivator to compare growth of the highest-expressing transformants to the control strain. Finally, if overexpression of the CAH1 gene increases growth in C. reinhardtii then this method could be applied to another algal species used in biofuel production, such as Chlorella vulgaris.

Demand for a more efficient, environmentally friendly fossil fuel alternative is increasing every day. Biofuels, derived from organic matter, could replace petroleum fuels. Specifically, algal biofuels have much promise. Algal biofuels surpass other types of biofuels because they: can be grown without reducing food stores, do not require arable land, have low fresh water requirements and can even grow in waste water. Carbon dioxide uptake, a major limiting factor in algal growth, is the focus of much algal biofuel research. Algae naturally evolved a process to increase intracellular carbon in a low carbon environments: the carbon concentrating mechanism (CCM). In this mechanism, there is one variety of CCM that enzymes converts CO₂ to HCO₃-, thus creating a concentration gradient and increasing carbon flow. Another enzyme transports HCO₃- across various cell membranes. The focus of this study is the CCM gene Low Carbon Inducible Protein 1 (LCI1). LCI1 codes for the LCI1 enzyme, which transports HCO₃- into the cell, thereby increasing carbon flow into the chloroplast where other enzymes convert HCO₃- back into CO₂ for carbon fixation. We aim to overexpress this gene using a ble 2a peptide insert, thereby increasing enzymatic activity to increase carbon accumulation and biofuel yield.

Biofuels are fuels that are sustainably produced from biological material and can be used to replace fossil fuels, which release pollutants and are in limited supply. Producing biofuels from algae will lessen fossil fuel demand, and the impact of greenhouse emissions. Algae are renewable, and can produce lipids that can be used to produce biofuels. Algae can be cultivated on wastewater not suitable for human consumption while absorbing CO2 from the atmosphere. Chlamydomonas reinhardtii is a unicellular photosynthetic green algae used for research in production of algal biofuels. Its fully mapped and largely annotated sequence, and relatively rapid growth makes this organism the best available model for biofuel research. This study focuses on the Carbonic Anhydrase 1 (CAH1) enzyme that converts CO2 into HCO3- in the cell periplasm. Our prediction is that over expression of this gene will increase the algal biomass by improving CO2 uptake. Using recombinant DNA technology, we ligated a synthesized CAH1 gene fragment into a C. reinhardtii expression vector and transformed into C. reinhardtii. None of the transformants expressed CAH1 protein so we modified the vector by ligating a ble-2A peptide fragment that permits easy selection for overexpressing transformants. One ligation product was verified by sequencing to have the ble-2A peptide fragment ligated in the correct orientation, and we are electroplating into C. reinhardtii. We will do western-blot analysis on transformants to determine transgenic protein expression. We will then use an algal multicultivator to compare growth of the highest-expressing transformants to the control strain. Finally, if overexpression of the CAH1 gene increases growth in C. reinhardtii then this method could be applied to another algal species used in biofuel production, such as Chlorella vulgaris.

Green algae are photosynthetic plant-like organisms that have great promise as a source of sustainable biofuels. Production of biofuels from algae is a sustainable alternative to fossil fuels and is potentially more economical than ethanol produced from corn, another alternative fuel source. Chlamydomonas reinhardtii, a single celled microalga, is the most widely used model organism for algal biofuel production research. Many tools are available for molecular genetic manipulation of C. reinhardtii, and it is easy to culture, making it an excellent platform for biotechnology research. The limiting factor for algal growth, carbon dioxide (CO), is the focus of much algal biofuels research. It is believed that certain enzymes involved in carbon dioxide uptake into cells for conversion to carbohydrates and eventually lipids, can be manipulated to improve photosynthesis and growth. The focus of this study are two genes that encode enzymes involved in CO2 uptake, CAH6 and CAH3. Carbonic anhydrase 6 (CAH6) converts CO2 into carbonate in the chloroplast stroma, increasing carbon flow into the pyrenoid, where CO2 fixation into carbohydrate takes place. Carbonic anhydrase 3 (CAH3) converts carbonate back into CO2 in the pyrenoid. Our prediction is that over expression of these enzymes should improve photosynthesis and thus cell growth. Utilizing recombinant DNA technology, we are generating vectors with CAH6 or CAH3 coding regions connected to ble gene sequence (encodes a selectable marker protein), with the hope of increasing expression of these proteins. We will electroporate these vectors into C. reinhardtii then use western-blot analysis to determine transgenic protein expression. Finally, we will use an algal multicultivator to observe and compare growth of our transformants to a control strain. If overexpression of CAH3 and CAH6 improves growth in C. reinhardtii, then our methods can be applied to other algal species, such as Chlorella vulagris, a biotechnology production organism.

High fossil fuel consumption and global warming concerns have encouraged exploration of renewable resources, including non-polar lipids produced by algae that can be converted into biodiesel. A major problem with algae as a source of biodiesel is their slow growth rate under conditions of high lipid production. We are testing the idea that overexpression of genes that increase carbon dioxide (CO2) uptake can increase metabolic function via the Calvin Cycle, leading to improved growth. In this research we are using the model green alga Chlamydomonas reinhardtii to test the effect of overexpressing a Carbon Concentrating Mechanism (CCM) component. The plasma membrane carbonate transporter LCI1 (Low Carbon Inducible) is responsible for inorganic carbon uptake into the cell. We first generated a nuclear expression vector that contained LCI1 coding sequence under the control of 5’ and 3’ regulatory sequences (HSP70A-RBSC3 hybrid 5’ UTR + promoter and RBCS2 3’ UTR), but we could not detect LCI1 protein in any of the transformants. To improve expression, we gene synthesized a bleomycin resistance gene fragment (ble) with a 3’ viral 2A peptide sequence and inserted it directly upstream of the LCI1 coding region in our first LCI1 vector. Bleomycin-resistant transformants should produce LCI1 since bleomycin expression is coupled to the expression of the downstream gene (LCI1). This construct will be transformed into C. reinhardtii and transformants will be tested via western blot for LCI1 protein accumulation. Expressing transformants will be analyzed for growth rates in an algal multi-cultivator to compare against the wild type. If this strategy is successful we will apply it to the commonly used industrial alga Chlorella vulgaris for use in biodiesel production.

Growing concerns over climate change are driving interest in development of renewable bioenergy to replace fossil fuels. Genetic manipulation of algae can make biofuel production more efficient. The photosynthetic green alga Chlamydomonas reinhardtii is a well-studied model organism that is easy to grow and manipulate at the molecular genetic level. This project focuses on a set of genes believed to be important for a carbon-concentrating mechanism (CCM) that acclimates algae to normal, CO2-limiting conditions. Carbonic anhydrases are components of the CCM that catalyze the interconversion of carbon dioxide and bicarbonate, and thereby make inorganic carbon more accessible to the cell. The purpose of this project is to increase the intracellular concentrations of CO2 in C. reinhardtii by overexpressing periplasmic and thylakoid membrane carbonic anhydrases, CAH1 and CAH3, respectively. C. reinhardtii CAH1 and CAH3 coding regions were synthesized with C. reinhardtii codon bias and epitope tags and the gene fragments were subcloned into expression vector pARG which contains the ARG7 gene required for arginine biosynthesis. We transformed the CAH1and CAH3 vectors into an arg7 mutant strain and selected several ARG survivors for western blot analysis to determine the expression of protein. We will select the best expressing lines for growth curve and dry weight analyzes to determine whether the transformants overexpressing CAH1 or CAH3 are able to grow faster than the wild-type C. reinhardtii strain. In future both genes could be expressed together. The next step will be to manipulate these methods for microalgae that naturally produce higher lipid levels than C. reinhardtii, such as Chlorella vulgaris.

Algae are plant-like organisms that can be used for sustainable production of biofuels and other commercially valuable products. Chlamydomonas reinhardtii, a single celled green alga, has been used as a model organism to research algal biofuel production, due to its sequenced genome, ability to be genetically manipulated and its fast growth rate. Carbon dioxide (CO2) is limiting for algal growth, so it is believed that certain enzymes that function in the Calvin cycle, which converts CO2 into carbohydrates, may be key targets for improving photosynthesis and growth. Fructose-bisphosphate aldolase (FBA) functions in the regeneration phase of the Calvin cycle, and overexpression of this enzyme in higher plants improves growth significantly. We are testing the idea that overexpressing FBA will also increase flux through the Calvin cycle in algae. Using recombinant DNA techniques, we have generated C. reinhardtii transformants that contain the coding region for C. reinhardtii FBA under the control of psbD and psbA 5’ and 3’ regulatory sequences, respectively, integrated into chloroplast genome. We are currently using western blot analysis to determine expression levels of FBA in these transformants. Next we will use an algal multicultivator to compare the growth rate of the best expressing transformants to that for the recipient (wild type) strain. If overexpression of FBA improves C. reinhardtii growth, we will apply these methods to other algae, such as the biotechnology production organism Chlorella.

Algal biofuels are an environmentally sustainable alternative to currently used and diminishing fossil fuels because they can be obtained directly from biomass derived from carbon dioxide (CO2) and sunlight. Increasing the photosynthetic activity of algae can increase their growth rate and biomass. One way to increase photosynthetic activity is to improve the Calvin cycle, a photosynthetic pathway of enzymatic reactions that convert CO2 and the energy of sunlight into sugars. Dual-function fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase (DFS) is a cyanobacterial enzyme that works to regenerate ribulose-1,5-bisphosphate (RuBP) in the regeneration phase of the Calvin cycle. Previously others found that overexpressing this cyanobacterial protein that possesses two Calvin cycle enzyme activities, could significantly improve plant biomass production. The goal of this project is to determine whether overexpressing this dual-function enzyme has the same effect in algae. We have generated C. reinhardtii transformants that contain the coding region for DFS under the control of psbD and psbA 5’ and 3’ regulatory sequences, respectively, integrated into the chloroplast genome. We analyzed the expression of this protein in transformants by western blot and found that the cyanobacterial protein is expressed. We will proceed by comparing the growth of these transformants with the wild type strain of C. reinhardtii. If expression of DFS leads to increased growth rate, then we would conclude that DFS carries out one or more rate-limiting steps in the Calvin cycle. If that is the case, we ultimately will overexpress DFS in a biotechnology production organism like Chlorella, in hopes of improving it as a biofuel-producing organism.

Photosynthetic green algae are found in environments that often contain low concentration of carbon dioxide (CO2) and bicarbonate ion (HCO3-) which are utilizable forms of inorganic carbon necessary for photosynthesis. Green algae overcome this stress disadvantage by inducing a carbon concentration mechanism (CCM) that includes carbonic anhydrases such as Cah1, Cah6 and inorganic carbon transporters such as Nar1.2. Chlamydomonas reinhardtii serves as an excellent model organism for the study of eukaryotic CCM. Carbon dioxide diffuses passively through cell membranes without any assistance from membrane bound proteins, but on the other hand bicarbonate ion needs transporters to facilitate its uptake across each membrane barrier. Nar1.2 is one of 6 nitrate/carbonate transporters (Nar1.1-Nar1.6), and is reputed to be the most important transporter with respect to CCM. Nar1.2 is localized to the chloroplast envelope and is believed to be part of the chloroplast inorganic carbon uptake system. Therefore over expression of Nar1.2 might significantly increase inorganic carbon uptake in Chlamydomonas reinhardtii and therefore increase photosynthesis and growth rate. To test this idea, Nar1.2 coding sequence was synthesized with specific enzymes restriction sites (BamH1 and Xho1) and was cloned into the pUC-ARG nuclear vector cut at those same sites. Next pUC-ARG-Nar1.2 will be transformed into Chlamydomonas reinhardtii using the glass bead transformation method. Transformants that express the transgenic Nar1.2 will be tested for growth rate compared to the wild type. If this strategy works, we will over express other CCM enzymes along with Nar1.2 in attempts to further increase growth rates.

One of the greatest challenges facing science and engineering is the development of efficient and renewable liquid fuels that are carbon neutral. Algal biofuels hold great promise in this regard because algae can photosynthesize and convert CO2 into high energy compounds such as lipids. However, low chloroplast CO2 levels generally limit algal growth. Atmospheric CO2 levels are very low (~0.04%), CO2 diffusion is 10,000 times slower in water than air, and cellular pH can make the balance between CO2 and its aqueous dissolved form (HCO3-) unfavorable.. To combat these factors, algae possess a carbon concentration mechanism (CCM) that manages the interconversion of CO2 and HCO3- inside different compartments of the cell. By manipulating the CCM, we may be able to successfully grow algae more efficiently. The goal of this project is to ultimately increase the intake and storage of carbon inside the model green algal species, Chlamydomonas reinhardtii. This study focuses on the gene that encodes Cah6, a CCM enzyme residing in the pyrenoid space of the chloroplast. We are attempting to over express Cah6 in order to increase the concentration of CO2 in the stroma of the chloroplast, increasing its availability for photosynthesis. We gene synthesized the C. reinhardtii CAH6 coding sequence, optimizing it for expression from the C. reinhardtii nuclear genome, and used standard cloning methods to insert it between flanking HSP70A and RBCS2 regulatory sequences in nuclear transformation vector pUC-ARG. After transforming into C. reinhardtii and selecting for Arg+ transformants, we will test for success by the transformants growth rates compared to standard C. Reinhardtii control growth rate. If successful, the over-expression of Cah6 allows for improved photosynthetic activity of C. Reinhardtii.

Algae hold great promise as a potential source for renewable liquid fuels, with most current research focused on increasing lipid content of natural or genetically engineered species. However, another important strategy for developing algae as a platform for biofuels production is to improve their growth rate. Our immediate goal is to determine if we can increase the growth rate of model organism Chlamydomonas reinhardtii through targeted genetic modifications, and our long-term goal is to apply successful modifications to other algae that naturally produce large amounts of lipids. Like many other phototrophs, Chlamydomonas has evolved a trait to survive under low carbon dioxide (CO2) levels in the atmosphere. The CO2 concentrating mechanism (CCM) works to accumulate inorganic carbon inside the chloroplase in the form of bicarbonate (HCO3) at a much higher concentration than atmospheric CO2 levels. We aim to repurpose the CCM to function with greater efficiency and under high CO2 conditions to stimulate rapid growth in Chlamydomonas. Within the CCM, we are targeting a carbonic anhydrase gene (CAH1) for overexpression. The CAH1 enzyme is located in the periplasmic space and is the first enzyme of the CCM to handle the conversion of CO2 to HCO3. To prepare our overxpression construct we cut our gene synthesized CAH1 coding sequence out of a pUC57 vector and litigated it into our nuclear expression vector pUC-ARG. Using the ARG7 gene as our selection marker, we will perform a nuclear transformation of Chlamydomonas then analyze transformants by western blot to determine if any overexpress CAH1. Any such transformants will subsequently be analyzed for growth rate and biomass production. If this effort is successful, the next step will be to generate a vector that overexpress additional CCM genes in hopes of improving growth rates even more.