Improved Part

What is this part? (Background)

The component BBa_K5008000 is an extension of BBa_K4161010, incorporating an additional DNA sequence that imparts new functionality to BBa_K4161010. Specifically, this component fuses a metal-binding protein (MBP) with CsgA. CsgA is a crucial subunit of the Escherichia coli peritrichous flagella involved in flagella assembly. Consequently, this component enables the presentation of MBP on the E. coli peritrichous flagella, thereby facilitating metal binding.

Figure 1. Peritrichous flagella schematic diagram of Escherichia coli. [1]

Why this part? (Ideal function)

CsgA (BBa_K4161010) has been widely used in the development of living biomaterials. It is involved in the formation of Escherichia coli flagella, and E. coli possesses multiple flagella. Therefore, by fusing CsgA with other proteins, it is possible to confer E. coli with the ability to display a wide range of proteins. For example, Figure 2 and the corresponding references demonstrate a case where CsgA is fused with the mineralizing peptide A7, imparting the ability for biomineralization on the resulting biomaterial.

Figure 2. The fusion expression of CsgA with the mineralizing peptide A7 imparts the capability to mineralize the biofilm. [2]

Here, we propose to fuse it with MBP, thereby conferring metal-binding functionality to Escherichia coli, which has broad applications. In this case, we have chosen a metal-binding protein specific to cadmium ions, allowing our engineered E. coli to possess cadmium ion-binding capability. We intend to utilize this strain of E. coli for the remediation of heavy metals in soil.

Figure 3. Our strategy for conferring cadmium ion-binding functionality to Escherichia coli [3]

Case from reference

In a previous study published by renowned synthetic biologist Timothy K. Lu, they systematically investigated the functionality of CsgA-MBP and observed the adsorption of numerous metal ions on the surface of Escherichia coli [2], as shown in Figure 4.

Figure 4. The microscopic images and detection results of metal ion adsorption by Escherichia coli

The work of this part

First, we utilized Colab's Alphafold2 [3] to model the structure of the fusion protein, as shown in Figure 5. It is evident from the image that our fusion protein did not alter the overall structure of CsgA, and our MBP was successfully displayed, indicating that this construct aligns with our design and theoretically should function as intended.

Figure 5. On the left is the official prediction of the CsgA protein by Alphafold2, while on the right is our prediction of the CsgA-MBP fusion protein using an online tool. In the fusion protein, the MBP portion is represented in orange. It can be observed that the fusion expression of MBP did not disrupt the structure of CsgA, and the MBP was successfully displayed.

In our experiment, we confirmed the binding of cadmium ions by using Congo Red staining. Congo Red can bind to fibrous substances, forming a red complex. In this case, it forms a red complex when bound to the flagella. Simultaneously, the cadmium ions we are binding exist in the form of cadmium sulfide (CdS), which further adsorbs Congo Red. Therefore, compared to BBa_K4161010, BBa_K5008000 exhibits a higher absorption at a wavelength of 450nm. Our experimental results align with this inference, indicating that BBa_K5008000 is a successfully modified part based on BBa_K4161010.

Figure 6. The experimental results showed that only the csgA (BBa_K4161010) group and the csgA plus MBP (BBa_K5008000) group were tested. The OD450/OD600 ratio was used to characterize the extent of Congo Red binding by the bacteria, with a higher ratio indicating more Congo Red binding. Considering that the quantity of bacterial flagella (composed of csgA) remained consistent, the increased binding of Congo Red was attributed to the presence of CdS, indicating that the E. coli expressing the BBa_K5008000 construct successfully bound CdS or cadmium ions. This demonstrates that the BBa_K5008000 construct, rather than the original BBa_K4161010 construct, conferred the ability for E. coli to bind metal ions.

To sum up, we have successfully modified BBa_K4161010 by adding metal-binding functionality to it. Our strategy not only applies to our specific project but also serves as a reference for other teams seeking to engineer the surface of Escherichia coli to confer new functionalities.