 Well, thank you for checking out this video highlight for our article in biotechnology and bioengineering. My name is Tushar Patel. I'm a graduate student under Dr. Scott Banta at Columbia University in the Department of Chemical Engineering. Our goal with this work was to create a wholesale biocadalyst in E. Coli for the hydration of CO2. We chose to do so to eliminate purification costs and also increase the recyclability of our catalysts. In order to create our wholesale biocadalysts, we used peri-plastic expression with carbonic anhydrous. We chose two different forms of carbonic anhydrase and two different leader peptides to create our catalysts. In gram-negative bacteria, such as E. Coli, the peri-plastic space is in between the inner and outer membranes of the bacteria. In order to localize this space, we chose the Pel-V and G3 leader peptides. Both are approximately 20 amino acids long and are fused to the n-terms of the protein in order to direct them into the peri-plastic. For the catalytic component, we chose carbonic anhydrase. Our carbonic anhydrases are zinc-bound metalloenzymes that are newly ubiquitous in nature. They are among the fastest known enzymes, with K-cats ranging from 10 to the fourth to 10 to the sixth per second. They are used in many natural processes, such as pH regulation in the blood and photosynthesis. Since there are so many forms of carbonic anhydrase, we selected two to begin with. The two we chose are Kav and K. Kav is a beta-type carbonic anhydrase that is one of the most thermostable types ever found. Kav is a gamma-type carbonic anhydrase and is used to oxidize an environment which the peri-plasm is. Both of these genes were optimized, synthesized, and cloned using standard molecular biology. To begin characterization, we first wanted to determine the location and amount of an enzyme per cell. In order to do so, we first treated the cells using osmotic shock. This separates the peri-plastic and cytosolid fractions of the cell. Then, running these samples on a western blood, we were able to determine where the enzyme is located. For all of our catalysts, we determined that most of the enzyme is located in the peri-plasm, as expected. Then, we can quantify these bands by using a standard curve generated using the Calmodule. By using this analysis, we found that LB actually generates more enzyme per cell than the G3-Pytidilis. The next step in characterization is to determine the kinetics of our whole cell biocatalyst as compared to the purified enzyme counterparts. In order to do so, we used stop-close spectroscopy, which allowed us to back out kinetic parameters for our enzymes as well as our whole cells. What we found is that there's about an order of magnitude loss in activity for the whole cell as compared to the purified enzymes. Now, this is expected since we have an ad-transport barrier from the other membrane of the cell. The trade-off to this loss in activity is that the enzymes are actually stabilized when immobilized in the whole cell. At 95 degrees, a smaller percentage of activity was lost to the whole cells than for their purified enzymes. We also saw that, operationally, the whole cell catalysts retain 100% of their original activity after 24 hours of use. As a potential application, we wanted to show that our whole cell biocatalyst could be used in carbon mineralization. Carbon mineralization is a carbon capture and storage technique, which combines carbonate ions with a divalent cation to precipitate as a carbonate cell. This has been touted as a much more permanent solution than geologic sequestration. For our experiments, we chose to precipitate calcium carbonate in the presence of our whole cell biocatalysts and also the relevant controls. By doing so, we saw a statistically significant increase in the amount of precipitation seen using our whole cell biocatalysts as opposed to the control of experiments. Hopefully in this video, we've shown you that we were able to successfully clone, express, and characterize the carbonic and hydrous-containing whole cell biocatalysts for CO2 hydration. For more information, please refer to our paper from the link below. At this time, I'd like to acknowledge my advisor, Dr. Scott Banta, our collaborator, Dr. Lisa Park, and my colleague Ed Swanson for all of their help. I'd also like to thank Arba E. and the Department of Energy for funding this work. Thank you.