Team:Nanjing-China/Description
Improvement of A Previously Existing Part BBa_J36836
As we all know, improving the function or characterization of previously existing parts in the part registry of iGEM is not only important for maintenance of the part registry, but essential for other teams to utilize the part properly as well.
Part BBa_J36836 encodes outer membrane protein (OmpA) of E. coli, which corresponds to a single transmembrane domain of OmpA. As far as we are concerned, this protein can be applied to the cell surface display system, which is to fix other proteins onto the surface of E.coli.
This year we extended the application of OmpA in the construction of our artificial PS system. In our project, a metal binding protein PbrR is fused with OmpA so as to induce precipitation of CdS nanoparticles on the surface of E.coli cells.
Protein PbrR is a special metal protein found in Cupriavidus metallidurans that specifically binds to Pb2+ ions. In realistic research we further examined that this protein also bears a high affinity to Cd2+ ions. We demonstrated in our project that when we fused PbrR with OmpA, the binding of PbrR with Cd2+ is greatly enhanced. (Figure 1).
Figure 1 The PbrR metal binding protein:
(left) structure of OmpA-PbrR fused protein; (right) metal specificity of PbrR
For organism M.thermoacetica, this kind of bacteria can produce S2- ions from cysteine and forms a higher sulfur concentration around the cell which then induces the precipitation of CdS nanoparticles when Cd2+ ions are added into the media. We assume that if we form a same local high concentration of Cd2+ with fused protein OmpA-PbrR on the outer cell membrane, we can also achieve a similar precipitation of CdS nanoparticles on to the walls of E.coli, the well model bacteria.
To confirm the capability of our CdS system based on OmpA-PbrR, we conducted the same photo-catalytic assay. Bacteria were divided into three groups. Bacteria were induced to express OmpA-PbrR protein and cultured with both Cd2+ and S2- in the experiment group. Groups that either lacked induced expression or necessary ions to build semiconductors were negative controls. We found that illumination resulted in a same increasing trend in experiment group (Figure 2). This confirmed the photo-catalytic capability of our PbrR-based precipitation of semiconductors.
Figure 2 photocatalytic reduction of MV by TiO2 and induced CdS nanoparticles
To conclude, we utilized the part BBa_J36836 and extended its application to the cell surface display system in E.coli. We successfully displayed a kind of metal binding protein PbrR to the surface of E.coli and identified enhanced function of PbrR after fusion with OmpA. To learn more details about our project design, please see the following project overview part.
Project Overview
Solar energy is the most widely used form of energy on earth, however it is also hard to harness. An improved photosynthesis can enable the sustainable production of chemicals from solar energy. Scientists have been making great efforts into the development of artificial photosynthetic (PS) systems.
Problems remain in two main sections for a PS system to harness solar energy and transform it into stable chemicals: one is the electron transmitter (like chlorophylls in plants), the other is downstream enzymes that capture the excited electrons (like light harvesting complex in plants). This year we plan to break through these two problems by new ways using synthetic biology.
For artificial PS systems, semiconductor nanoparticles such as CdS and TiO2 can mimic pigment chlorophylls to provide excited electrons under illumination. Traditional ways require both chemically synthesized nanoparticles which are generally expensive as well as bio-incompatible and purified enzymes extracted from cell homogenates.
Exactly these years have seen a combined method using special organism Moorella thermoacetica to naturally form CdS nanoparticles on the cell walls with its unique metabolism, however this approach is really hard to be applied onto other species. This year we work against this obstacle with an induced similar semiconductor formation on the cells of model species E.coli using special metal-binding protein PbrR and OmpA surface display machinery.
For the second part we found that many downstream enzymes including hydrogenase and nitrogenase are generally oxygen sensitive. This year we come up with the idea of silicon encapsulation to counter oxygen sensitivity. When chlorella is wrapped into microballs using polymerized silicon materials, they create a gas gradient where outer cells consumes oxygen, leaving an anaerobic condition in the core which protects the fragile enzymes within. We plan to test if this principle also works with E.coli.
At last we will combine our surface-displayed solar driven system with downstream enzymes in designed silicon coat. We achieve in air hydrogen production using E.coli hydrogenase 1 as our proof. This new light-driven system on model organism E.coli is of general applications which can also extend to other enzymes and more species including the very-important engineering vessel yeast and bacillus.
Artificial Photosynthetic System
Figure 3 plant photosynthetic system and artificial photosynthetic system
For photosynthetic systems in plants (Figure 3 left), photons excite the electrons from pigment chlorophyll molecules located on the membrane of thylakoids. The electrons are then captured by pheophytin and passed down to electron transport chain that ends in the reduction force equivalent NADPH. The chlorophyll ultimately regains its lost electrons from water, releasing oxygen in photolysis.
The artificial PS system is composed of similar components (Figure 3 right). The pigment chlorophyll is changed into semiconductors, which also provides excited electrons under illumination. The lost electrons from semiconductors could be provided by sacrificial electron donors and we employ ascorbic acid in this project. The reduction force equivalent will then be hydrogen produced from hydrogenase instead of the unstable NADPH.
The exact advantage of artificial PS systems over natural ones is that they have a wider range of application in different enzymes and non-photosynthetic species. However current methods for the construction of artificial PS system either required expensive semiconductor particles, purified enzyme or certain species with unique metabolism. Thus this year we introduce the new approach using PbrR on the model organism E.coli.
Silicon Encapsulation
Experiments concerning oxygen-sensitive enzymes are generally done in glove box due to its oxygen sensitivity, which requires strict and inconvenient anaerobic operations. This year we propose the idea of silicon encapsulation to overcome all these trouble.
Figure 4 encapsulation from LbL assembly to create SFD structure
The principle is “spatial functional differentiation” (SFD), which has been tested out in chlorella. Chlorellas only produce hydrogen when oxygen is removed under natural conditions because of oxygen sensitivity of its hydrogenase. Research has found that silicification-induced chlorella microballs lead to stable aerobic production of hydrogen. This aggregate structures a core-shell complex in which the shell consumes the oxygen with respiration and to some extent acts as a barrier blocking the diffusion of air oxygen. Thus the core gained hydrogen production capabilities in an isolated microenvironment with oxygen exploited. The same idea may work with E.coli (Figure 4).
Our induced aggregation is achieved through “Layer-by-Layer” self-assembly. On the basis that E.coli cell membrane is negatively charged, a cationic polyelectrolytes: poly (diallyldimethylammonium chloride) or PDADMA construct the first layer to wrap cell aggregate by attractive electrostatic interactions. Then a negatively charged layer: anionic polyelectrolyte sodium polystyrene sulfonate (PSS) is deposited onto the first one according to the same principle. To assure that enough cell number and stability for encapsulation coat, this process is repeated to form a PDADMAC/PSS (10/11) multilayer ends with PDADMAC (11 layers). The last step surrounds the coat with negatively charged silicic acid and eventually forms silica-encapsulated E.coli cell group (Figure 4).
Hydrogenase
Serving as the enzyme capturing light induced electrons and being generally oxygen sensitive, hydrogenase is our target enzyme. Hydrogenase can be defined as the enzyme that catalyzes the reversible oxidization of hydrogen which produces hydrogen out of protons if electrons are provided.
Figure 5 three classes of hydrogenases
Hydrogenase widely found among microbes. There naturally exist three classes of hydrogenases in all, divided by their catalytic center: the [Fe-Fe], [Ni-Fe], and [Fe] hydrogenases (Figure 5). The di-iron hydrogenase bears a very instablity and [Fe] hydrogenase is only recently found in a small group archaea. Thus we choose [Ni-Fe] hydrogenase as our target enzyme.
Figure 6 three [Ni-Fe] hydrogenases found in E.coli: EcHyd-1(left), EcHyd-2(middle), EcHyd-3(right)
There are in all three [Ni-Fe] hydrogenases found in E.coli: EcHyd-1, 2 and 3 (Figure 6). Hyd3 is too complex to design and Hyd2 is less active. Hyd1 suits our need best. So we choose Hyd1 as our target enzyme in E.coli.
Figure 7 structure of operon hyaABCDEF
EcHyd-1 in encoded by a six-gene operon hyaABCDEF on the genome of E.coli (Figure 7). The first two genes hyaA and hyaB encode the small and large subunit for the enzyme and hyaCDEF is of important function in the enzyme’s maturation. All six genes will also be our Parts this year.
Our Design
Figure 8 the induced precipitation of CdS nanoparticles using fused protein OmpA-PbrR
To conclude our light-driven system, we first induce the precipitation of CdS nanoparticles on the cell membrane. Two plasmids encoding the building block OmpA-PbrR and the enzyme HyaABCDEF are co-transformed into E.coli strain. Arabinose is used to induce the expression of OmpA-PbrR fused protein. Then when Cd2+ is added into the media, the ions specifically binds to PbrR leading to a locally high concentration of Cd2+ ions. At last when S2- ions are added into the media, E.coli cells form in situ CdS nanoparticles on the cell membrane because of this local high concentration.
If our design proves to be successful, we can also expand the applications of our artificial photosynthetic system into other model organisms such as B. subtilis and yeast simply by replacing OmpA with TasA/CotC in B. subtilis and GCW21 in yeast, as all these proteins are cell surface display proteins (Figure 9). Besides, we can also expand our encapsulation system into other oxygen-intolerant enzymes.
Figure 9. Expansion of our artificial photosynthetic system into other organisms.
Please go to our Proof-of-Concept page if you want to know if our amazing design really works!
References
[1] Wei Wei, Xiangzhi Liu, Peiqing Sun, et al. Simple Whole-Cell Biodetection and Bioremediation of Heavy Metals. Based on an Engineered Lead-Specific Operon[J].Environ. Sci. Technol. 2014, 48, 3363−3371
[2] Kelsey K. Sakimoto, Andrew Barnabas Wong, Peidong Yang. Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production[J].Science,2016.1.1,351(6268), 74-77
[3] Yuki Honda, Hidehisa Hagiwara, Shintaro Ida, and Tatsumi Ishihara. Application to Photocatalytic H2 Production of a Whole-Cell Reaction by Recombinant Escherichia coli Cells Expressing [FeFe]-Hydrogenase and Maturases Genes[J].Angew. Chem. Int. Ed.2016, 55, 1-5
[4] Davide Zannoni, Roberto De philippis. Microbial BioEnergy: Hydrogen Production[M].Springer:Dordrecht Heidelberg New York London,2014.ISBN 978-94-017-8553-2
[5] Katherine A. Brown, Derek F. Harris, Molly B. Wilker, et al. Light-driven dinitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid[J].Science,2016.4.22,352(6284), 448-450
[6] Wei Xiong, Xiaohong Zhao, Genxing Zhu, et al. Silicification-Induced Cell Aggregation for the Sustainable Production of H2 under Aerobic Conditions[J].Angew. Chem. Int. Ed. 2015, 54, 11961-11965
[7] Sung Ho Yang, Kyung-Bok Lee, Bokyung Kong, et al. Biomimetic Encapsulation of Individual Cells with Silica[J].Angew. Chem. Int. Ed. 2009, 48, 9160-9163
[8] Lucia Forzi, R. Gary Sawers. Maturation of [NiFe]-hydrogenases in Escherichia coli[J]. Biometals, 2007, 20, 565-578
[9] Paul C. Jordan1, Dustin P. Patterson, Kendall N. Saboda, et al. Self-assembling biomolecular catalysts for hydrogen production[J].Nature Chemistry, 2015.12.21
[10] Suzannah V. Hexter, Min-Wen Chung, Kylie A. Vincent,and Fraser A. Armstrong. Unusual Reaction of [NiFe]-Hydrogenases with Cyanide[J].Journal of the American Chemical Society, 2014, 136, 10470-10477
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