Improvement by Squirrel-Shanghai 2023

CsgA_MBP

The component BBa_K5008000 adds an additional DNA sequence based on BBa_K4161010. This DNA sequence will interact with BBa_K4161010 and give BBa_K4161010 new functions. Specifically, this part is fused with a metal binding protein (MBP) based on CsgA. At the same time, CsgA is a very important subunit of the E. coli peritrichous flagellum and participates in the assembly of the flagellum. Therefore, our part can enable the E. coli peritrichous flagellum to present MBP and play a metal-binding function.

Figure 1. Schematic diagram of E. coli peritrichous flagellum[1]

Ideal function

CsgA (BBa_K4161010) is widely used in the development of living biological materials. It participates in the formation of E. coli flagella, and E. coli has many flagella. Therefore, after CsgA is fused and expressed with other proteins, E. coli can be given the function of broadly presenting proteins. For example, the cases in Figure 2 and corresponding references demonstrate the fusion of CsgA and the mineralizing short peptide A7 to confer biofilm mineralization ability.

Figure 2. Fusion expression of CsgA and mineralization short peptide A7 gives biofilm the ability to mineralize [2]

Here we propose to fuse it to express MBP, so that E. coli has a metal-binding function, which has a wide range of applications. Here, we selected metal-binding proteins for cadmium ions, thereby conferring cadmium ion-binding functionality to our engineered E. coli . We hope to use this strain of E. coli to treat heavy metals in soil.

Figure 3. Our strategy to confer cadmium ion binding functionality to E. coli[3]

Cases from references

In a previous study published by Timothy K. Lu, a well-known researcher in the field of synthetic biology, they systematically tested the working condition of CsgA-MBP, and it can be seen that many metal ions are adsorbed on the surface of E. coli [2], as shown in Figure 4 shown.

Figure 4. Microscope and detection results of metal ions adsorbed by E. coli.

The work of this part

First, we used Colab's Alphafold2 [3] to model the fusion protein structure, as shown in Figure 5. It can be seen very clearly that our fusion protein does not change the overall structure of CsgA, and our MBP was successfully demonstrated, indicating that this structure conforms to our design and can work in theory.

Figure 5. The left side is the CsgA protein officially predicted by Alphafold2, and the right side is the CsgA-MBP fusion protein predicted by us using online tools. Among them, the MBP part is indicated in orange. It can be seen that the fusion expression of MBP does not destroy the structure of CsgA, and MBP is successfully displayed.

In our experiments, we confirmed its binding to cadmium ions by Congo red staining. Congo red can combine with fibrous substances to form a red complex, that is, it combines with flagella to form a red complex. At the same time, the cadmium ions we combine exist in the form of cadmium sulfide CdS, which will further adsorb Congo red, so BBa_K5008000 and BBa_K4161010 In comparison, the former will have greater absorption at the 450nm wavelength. Our experiments are consistent with this inference, indicating that BBa_K5008000 is a successfully modified part based on BBa_K4161010.

Figure 6. Experimental results. Only csgA is the BBa_K4161010 group, and csgA plus MBP is the BBa_K5008000 component. OD450/OD600 is used to characterize the degree of Congo red bound by bacteria. The higher it is, the more Congo red is bound. Considering that the number of bacterial flagella (composed of csgA) is consistent, the multi-binding Congo red is caused by CdS, indicating that E. coli expressing the element BBa_K5008000 successfully binds CdS or cadmium ions, indicating that this element rather than the initial element BBa_K4161010 is endowed to E. coli The binding function of metal ions.

To sum up, we have successfully modified BBa_K4161010 and added the metal bonding function on it. Our strategy can not only be used in our project, but also provide a reference for other teams that need to modify the surface of E. coli to give it new functions.

References

[1] https://www.uniprot.org/uniprotkb/P28307/entry

[2] DOI: 10.1126/sciadv.abm7665

[3] DOI: 10.1021/acssynbio.9b00235

[4] Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: Making protein folding accessible to all. Nature Methods, 2022

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 371
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 371
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 371
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 371
1000
COMPATIBLE WITH RFC[1000]

Source