Matching Items (5)

134124-Thumbnail Image.png

Creating and Characterizing a PsaC-HydA1 Fusion in Chlamydomonas reinhardtii

Description

There is an ever-increasing need in the world to develop a source of fuel that is clean, renewable and feasible in terms of production and implementation. Hydrogen gas presents a possible solution to these energy needs, particularly if given a

There is an ever-increasing need in the world to develop a source of fuel that is clean, renewable and feasible in terms of production and implementation. Hydrogen gas presents a possible solution to these energy needs, particularly if given a way to produce hydrogen gas efficiently. Biological hydrogen (biohydrogen) production presents a potential way to do just this. It is known that hydrogenases are active in wild-type algal photosynthesis pathways but are only active in anoxic environments, where they serve as electron sinks and compete poorly for electrons from photosystem I. To circumvent these issues, a psaC-hydA1 fusion gene was designed and incorporated into a plasmid that was then used to transform hydrogenase-free Chlamydomonas reinhardtii mutants. Results obtained suggest that the psaC-hydA1 gene completely replaced the wild-type psaC gene in the chloroplast genome and the fusion was expressed in the algal cells. Western blotting verified the presence of the HydA1-PsaC fusion proteins in the transformed cells, P700 photobleaching suggested the normal assembly of FA/FB clusters in PsaC-HydA1, and PSII fluorescence data suggested that HydA1 protein limited photosynthetic electron transport flow in the fusion. Hydrogen production was measured in dark, high light, and under maximal reducing conditions. In all conditions, the wild-type algal strain (with a normal PsaC protein) exhibited higher rates of hydrogen production in the light over 2 hours than the WT strain, though both strains produced similar rates in the dark.

Contributors

Agent

Created

Date Created
2017-12

152182-Thumbnail Image.png

Modification of electron transfer proteins in the Chlamydomonas reinhardtii chloroplast for alternative fuel development

Description

There is a critical need for the development of clean and efficient energy sources. Hydrogen is being explored as a viable alternative to fuels in current use, many of which have limited availability and detrimental byproducts. Biological photo-production of H2

There is a critical need for the development of clean and efficient energy sources. Hydrogen is being explored as a viable alternative to fuels in current use, many of which have limited availability and detrimental byproducts. Biological photo-production of H2 could provide a potential energy source directly manufactured from water and sunlight. As a part of the photosynthetic electron transport chain (PETC) of the green algae Chlamydomonas reinhardtii, water is split via Photosystem II (PSII) and the electrons flow through a series of electron transfer cofactors in cytochrome b6f, plastocyanin and Photosystem I (PSI). The terminal electron acceptor of PSI is ferredoxin, from which electrons may be used to reduce NADP+ for metabolic purposes. Concomitant production of a H+ gradient allows production of energy for the cell. Under certain conditions and using the endogenous hydrogenase, excess protons and electrons from ferredoxin may be converted to molecular hydrogen. In this work it is demonstrated both that certain mutations near the quinone electron transfer cofactor in PSI can speed up electron transfer through the PETC, and also that a native [FeFe]-hydrogenase can be expressed in the C. reinhardtii chloroplast. Taken together, these research findings form the foundation for the design of a PSI-hydrogenase fusion for the direct and continuous photo-production of hydrogen in vivo.

Contributors

Agent

Created

Date Created
2013

132373-Thumbnail Image.png

Optimizing the Production of Algal Biohydrogen

Description

The oxygen sensitivity of hydrogenase is a large barrier in maximizing the efficiency of algal hydrogen production, despite recent efforts aimed at rewiring photosynthesis. This project focuses on the role of photosystem II (PSII) in extended hydrogen production by cells

The oxygen sensitivity of hydrogenase is a large barrier in maximizing the efficiency of algal hydrogen production, despite recent efforts aimed at rewiring photosynthesis. This project focuses on the role of photosystem II (PSII) in extended hydrogen production by cells expressing the PSI-HydA1 chimera, with the goal of optimizing continuous production of photobiohydrogen in the green alga, Chlamydomonas reinhardtii. Experiments utilizing an artificial PSII electron
Therefore, it can be concluded that downstream processes are limiting the electron flow to the hydrogenase. It was also shown that the use of a PSII inhibitor, 3-(3,4-dichlorophenyl)-1,1- dimethylurea (DCMU), at sub-saturating concentrations under light exposure during growth temporarily improves the duration of the H2 evolution phase. The maximal hydrogen production rate was found to be approximately 32 nmol h-1 (µg Chl)-1. Although downregulation of PSII activity with DCMU improves the long-term hydrogen production, future experiments must be focused on improving oxygen tolerance of the hydrogenase as a means for higher hydrogen yields.

Contributors

Agent

Created

Date Created
2019-05

149368-Thumbnail Image.png

Isolation, purification and characterization of photosynthetic membrane proteins from Galdieria sulphuraria and Chlamydomonas reinhardtii

Description

In oxygenic photosynthesis, Photosystem I (PSI) and Photosystem II (PSII) are two transmembrane protein complexes that catalyze the main step of energy conversion; the light induced charge separation that drives an electron transfer reaction across the thylakoid membrane. Current knowledge

In oxygenic photosynthesis, Photosystem I (PSI) and Photosystem II (PSII) are two transmembrane protein complexes that catalyze the main step of energy conversion; the light induced charge separation that drives an electron transfer reaction across the thylakoid membrane. Current knowledge of the structure of PSI and PSII is based on three structures: PSI and PSII from the thermophilic cyanobacterium Thermosynechococcus elonagatus and the PSI/light harvesting complex I (PSI-LHCI) of the plant, Pisum sativum. To improve the knowledge of these important membrane protein complexes from a wider spectrum of photosynthetic organisms, photosynthetic apparatus of the thermo-acidophilic red alga, Galdieria sulphuraria and the green alga, Chlamydomonas reinhardtii were studied. Galdieria sulphuraria grows in extreme habitats such as hot sulfur springs with pH values from 0 to 4 and temperatures up to 56°C. In this study, both membrane protein complexes, PSI and PSII were isolated from this organism and characterized. Ultra-fast fluorescence spectroscopy and electron microscopy studies of PSI-LHCI supercomplexes illustrate how this organism has adapted to low light environmental conditions by tightly coupling PSI and LHC, which have not been observed in any organism so far. This result highlights the importance of structure-function relationships in different ecosystems. Galdieria sulphuraria PSII was used as a model protein to show the amenability of integral membrane proteins to top-down mass spectrometry. G.sulphuraria PSII has been characterized with unprecedented detail with identification of post translational modification of all the PSII subunits. This study is a technology advancement paving the way for the usage of top-down mass spectrometry for characterization of other large integral membrane proteins. The green alga, Chlamydomonas reinhardtii is widely used as a model for eukaryotic photosynthesis and results from this organism can be extrapolated to other eukaryotes, especially agricultural crops. Structural and functional studies on the PSI-LHCI complex of C.reinhardtii grown under high salt conditions were studied using ultra-fast fluorescence spectroscopy, circular dichroism and MALDI-TOF. Results revealed that pigment-pigment interactions in light harvesting complexes are disrupted and the acceptor side (ferredoxin docking side) is damaged under high salt conditions.

Contributors

Agent

Created

Date Created
2010

165656-Thumbnail Image.png

Bioengineering for Cleaner Water: iGEM at ASU

Description

Arsenic contamination in groundwater is a serious problem both in local Arizonan communities and abroad: prolonged exposure to arsenic contamination can cause cancer, vascular damage, and liver failure. This project aims to engineer the microalgae Chlamydomonas reinhardtii to sequester arsenic

Arsenic contamination in groundwater is a serious problem both in local Arizonan communities and abroad: prolonged exposure to arsenic contamination can cause cancer, vascular damage, and liver failure. This project aims to engineer the microalgae Chlamydomonas reinhardtii to sequester arsenic out of water. Metallothionein, arsenate reductase, and ferritin were integrated into the microalgae via the pASapI plasmid. The plasmid rescues function of the photosystem II gene, leveraging the ability to photosynthesize as a selective trait. Metallothionein and ferritin bind the two most common forms of arsenic: arsenite and arsenate, respectively. Arsenate reductase catalyzes the reduction of arsenate to arsenite, allowing for the ultimate sequestration of the toxic metal to occur in the chloroplast. The algae was transformed using a biolistic device, to create three mutant strains, expressing Metallothionein (MT), Arsenate Reductase (ArsC)-HA, and MT-6xHIS plasmids respectively. When testing the fluorescence output of these three strains, they showed a maximum quantum yield of photosystem II comparable to that of the wildtype algae, indicating that the rescue gene had been incorporated into the chloroplast genome properly. Strains were exposed to arsenic-containing media at 50ppb and 500 ppb for 48 and 72 hours to determine the arsenic sequestration rate. Arsenic concentration in the supernatant was measured using ICP-MS analysis and sequestration rate was calculated in terms of arsenic concentration per fold growth of algae. The normalized arsenic sequestration rates of tagged protein expressing strains at 50 ppb were significantly higher than wildtype.

Contributors

Agent

Created

Date Created
2022-05