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Production, Purification, and Characterization of recombinant β-glucosidase F57Y

Elena Flores Cabrera and Fabiana Pena Medina

Saddleback College Department of Biological Sciences

Saddleback College, Mission Viejo, CA and UC Davis, Davis, CA

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Abstract

The purpose of the experiment was to work with a larger network of labs alongside UC Davis, in an effort to harness the power of crowdsourcing in science research. The experiment aimed to identify a specific plasmid whose mutations have led to a change in enzyme activity. In order to do this, competent BLR21 E. coli cells were transformed with a plasmid which contained a mutated sequence of the Glucosidase gene. This was done by using the Design-to-Data workflow developed by the Siegel Lab at UC Davis. To begin, BRL21 E. coli cells were transformed with the plasmid F57Y. Subsequently, protein expression was induced using IPTG. Afterward, the BLR cells were grown overnight, to be used in the next step, Immobilized Metal Affinity Chromatography. This step uses the histidine-tagged protein which interacts with nickel beads in a chromatography column for protein purification. A kinetic assay was used to compare activity of the enzyme against the wild-type. A final step of SDS-PAGE allowed for analysis of protein purity.

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BLR21

TRANSFORMATION

BACTERIAL

GROWTH

PROTEIN

PURIFICATION

Materials and Methods

Transformed Cells Growth and Expression Lysed Cell Pellet Affinity Chromatography Purified Protein

To begin, we transformed BLR cells, that have an e coli strain and no lon protease. Also has to be deficient in outer membrane protease OmpT. The lack of the ion protease and protease ompt reduces degradation of heterologous proteins expressed in the cells.

Cells transformed with heat shock, they will grow in a agar plate with kanamycin (antibiotic).

Using IPTG we will induce high levels of proteins.

Lysed cells were spun into a pellet and column containing nickel will be used to extract the his-tagged BgIB.

E. coli cells have the ability to grow on media containing the antibiotic kanamycin, which is why they were able to grow during part 1.

During transformation, we selected an isolated colony using a clean pipet tip and placed it into cen tube

Transformed E. coli B strain cells, which have a unique gene for kanamycin resistance.

Transformed cells will multiply undergo induce protection expression of the BgIB mutant enzyme with IPTG.

The cells were lysed and prepared the mutant enzyme for purification by using immobilized metal affinity chromatography.

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KINETIC ASSAY &

SDS-PAGE

Materials and Methods

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Results

Figure 2. Analysis of the purity of the protein using SDS-PAGE.

Figure 1. Comparison of several mutants against the wild type control. Mutant 22 and mutant 23 correlated with the expected result; substrate concentration increases as the velocity of reaction also increases.

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Conclusion/Future Experiments

https://researchoutreach.org/articles/cellulose-nanodefects-key-biofuels-biomaterials-future/

due to its sugary structure, cellulose has the potential to be used for biofuel production, which requires the depolymerisation and isolation of smaller sugar units that could then be transformed into fuel. This potential is currently untapped, despite rigorous research over the past decades on physical, chemical, and biological methods that could be used to break down cellulose structure.

https://www.energy.gov/articles/6-new-things-happening-biofuels#:~:text=A%20brand%20new%20ARPE%2DE,new%20strains%20of%20the%20plant.

Sorghum, a plant with high sugar content ideal for producing biofuel.

Because of its sugary structure, cellulose has the potential to be used in biofuel production. Cellulosic biofuels are able to offset more gasoline than grains. There are so many great environmental benefits of using cellulose for biofuels. But that also depends on what kind of cellulose plants are used which is why it is important to understand BgIB activity and conducting further testing with other mutants to be able to find the best alternatives. https://lter.kbs.msu.edu/wp-content/uploads/2012/06/sustainability-of-cellulosic-biofuels.pdf

https://www.google.com/search?q=why+are+biofuels+important+youtube&sca_esv=2f61a0c390a4d1ff&ei=1NgxZv_sCI7QkPIP8ZW2-As&ved=0ahUKEwj_pqzW4euFAxUOKEQIHfGKDb8Q4dUDCBE&uact=5&oq=why+are+biofuels+important+youtube&gs_lp=Egxnd3Mtd2l6LXNlcnAiIndoeSBhcmUgYmlvZnVlbHMgaW1wb3J0YW50IHlvdXR1YmUyBRAhGKABSK0KUPQCWJ0JcAF4AZABAJgBTKABsgSqAQE4uAEDyAEA-AEBmAIJoALUBMICChAAGLADGNYEGEfCAgsQABiABBiRAhiKBcICBhAAGBYYHsICCxAAGIAEGIYDGIoFwgIHEAAYgAQYDcICCBAAGIAEGKIEwgIFECEYnwWYAwCIBgGQBgiSBwE5oAeRKA&sclient=gws-wiz-serp#kpvalbx=_19gxZq2pK-HLkPIPj8-P8AI_43

Biofuels are fuels that are created from organic material biomass either directly or indirectly. Biofuels supply with bioenergy that take care about 10 percent of the worlds energy needs. Can be a liquid, solid or gas. Biofuels can be extracted from waste, wood or crops.

Biofuels are used for cooking, heating and electricity. 2nd biofuels used for motor vehicles, large scale manufacturing.

Plant matter made up of cellulose, and can produce ethanol. NOTE: the use of liquid biofuels has disadvantages such as increase cost, industrial pollution, global warming. Biofuels are in high demand.

Understanding the enzyme activity of BgIB is important to the environment because it is the last enzyme needed in a reaction cascade in order to break down cellulose thus having a great impact on biofuels. Its structure is also important to more than one biological system.

Biofuels apply to liquid fuels and blending components produced from biomass materials called feedstocks. Biofuels may also include methane production from landfill gas and biogas and hydrogen produced from renewable resources. https://www.eia.gov/energyexplained/biofuels/#:~:text=The%20term%20biofuels%20usually%20applies,hydrogen%20produced%20from%20renewable%20resources.

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References/Literature Cited

Singh, G., Verma, A. K., & Kumar, V. (2016). Catalytic properties, functional attributes and industrial applications of β-glucosidases. 3 Biotech, 6(1), 3. https://doi.org/10.1007/s13205-015-0328-z

Mariano, D., Pantuza, N., Santos, L. H., Rocha, R. E. O., de Lima, L. H. F., Bleicher, L., & de Melo-Minardi, R. C. (2020). Glutantβase: a database for improving the rational design of glucose-tolerant β-glucosidases. BMC molecular and cell biology, 21(1), 50. https://doi.org/10.1186/s12860-020-00293-y

Srivastava, N., Rathour, R., Jha, S., Pandey, K., Srivastava, M., Thakur, V. K., Sengar, R. S., Gupta, V. K., Mazumder, P. B., Khan, A. F., & Mishra, P. K. (2019). Microbial Beta Glucosidase Enzymes: Recent Advances in Biomass Conversation for Biofuels Application. Biomolecules, 9(6), 220. https://doi.org/10.3390/biom9060220

Huang, P., Chu, S. K. S., Frizzo, H. N., Connolly, M. P., Caster, R. W., & Siegel, J. B. (2020). Evaluating Protein Engineering Thermostability Prediction Tools Using an Independently Generated Dataset. ACS omega, 5(12), 6487–6493. https://doi.org/10.1021/acsomega.9b04105

Kathryn G. Guggenheim, Lauren M. Crawford, Francesca Paradisi, Selina C. Wang, and Justin B. Siegel ACS Omega 2018 3 (11), 15754-15762 DOI: 10.1021/acsomega.8b02169

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Acknowledgments

We would like to thank the Saddleback College Department of Biological Sciences, our Bio 3C: Biochemistry and Molecular Biology cohort, Dr. Monica Friedrich, and the UC Davis Siegel Lab for the resources and support needed to accomplish this experiment. We would also like to thank NSF, ThermoFisher, Rosetta Commons, Justin Siegel, and Ashley Vater.