Overview

At the beginning of our project, we read about many works from our predecessors. From their descriptions, we were able to gain a lot of information. At the same time, a lot of inspiration and insight were got and the proven knowledge was applied to our topic to make our design more relevant to the topic, which advanced our project greatly. We believe that making a difference for the future iGEMers must be one of our goals. We expect that the contributions we made will inspire the other teams. All of this would realize one produces two and two produce more and make the iGEM community more colorful.

Contribution (Bronze Criterion #4)

Cytolysin A (ClyA) is a pore-forming toxin that is produced by some bacteria from the Enterobacteriaceae family (1). And INPNC is a truncated form of ice nucleation protein (INP) consisting of N- and C-terminal domains (2). Both two parts were first registered in 2009 and used as membrane proteins to display cargo proteins on the cell surface (Fig. 1a).

For the Contribution, we completed the experimental characterization of the previous parts (BBa_K257002 and BBa_K265008) and added new data of them to the corresponding BioBricks.

We constructed four gene circuits in E. coli BL21(DE3) to implement the characterization experiments. All of these may be helpful to other teams. We hope it will make contribution to the iGEM community.

Fig. 1 The illustration and characterization of the surface display systems. a Displaying proteins illustration for the two systems. The left is for BBa_K4195135 and the right is for BBa_K4195136. b Fluorescence intensity/OD₆₀₀ of E. coli whether express the anchor protein or not.

The surface display system is characterized by immunofluorescence (IF). The culture was incubated overnight. Then the FITC-labeled anti-His-Tag antibody was used to target the His-tag (6×His) displayed via ClyA and INPNC, followed by measuring the fluorescence intensity (λ_Ex = 492 nm, λ_Ex = 518 nm) and OD₆₀₀ of the culture.

The results showed that the ratio of fluorescence intensity to OD₆₀₀ of positive control (bacteria harboring BBa_K4195135 or BBa_K4195136) is higher than that of negative control (bacteria harboring BBa_K4195119) (Fig. 1b), which indicated that these two surface display systems can work well. The further use and characterization results of these two anchor proteins to display several cargo proteins in our project could be found in our Design and Results pages.

Reference

1. K. Murase, Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. Toxins (Basel) 14, 78 (2022).
2. E. van Bloois, R. T. Winter, H. Kolmar, M. W. Fraaije, Decorating microbes: surface display of proteins on Escherichia coli. Trends Biotechnol. 29, 79-86 (2011).