Part:BBa_K4968011
CsgA-AG4-CsgC-CsgD-CsgE-CsgF-CsgG
This composite part consist of CsgA-AG4 (BBa_K4968008), CsgC-CsgD(BBa_K4968009), and CsgE-CsgF-CsgG(BBa_K49680010).
Designed composite part to compensate for the missing curli operon in JF1. Aims to express AG4 protein, secrete it extracellularly, and adsorb silver ions for validation. We intend to use Congo Red Dye to stain amyloid (mainly CsgA), ICP-MS to verify the functionality of the fusion protein in adsorbing silver ions, and SEM to further verify and observe the expression of the CsgA-AG4 fusion protein.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1958
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1958
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1958
Illegal BglII site found at 198 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1958
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1958
Illegal NgoMIV site found at 58
Illegal AgeI site found at 2269 - 1000COMPATIBLE WITH RFC[1000]
Usage & biology
The curli operon, which plays a crucial role in curli fiber assembly, was entirely removed from the genome of the JF1 strain. This genetic manipulation necessitates the creation of a custom plasmid containing all components of the curli operon, excluding CsgB, as well as our target protein AG4. This plasmid aims to restore curli fiber assembly capabilities to JF1 and serves as a platform to investigate AG4 functionality.
The design of the CsgA-AG4-CsgC-CsgD-CsgE-CsgF-CsgG plasmid involves the construction of a genetic cassette capable of directing the synthesis of the curli fiber components, with AG4 as the added feature. AG4, in this context, serves as a protein with potential applications in adsorbing valuable silver ions from the environment (Seker and Demir, 2011). CsgC and CsgD each have distinct roles in the regulation of curli fiber assembly and biofilm formation. According to Evans et al. (2015), CsgC is involved in preventing the premature polymerization of curli subunits, ensuring that the assembly occurs outside the bacterial cell where it's intended. This chaperone-like protein binds to curli subunits and prevents their aggregation within the cell, enabling controlled and organized assembly once they are transported outside (Taylor et al., 2016). CsgD functions as a master regulator that orchestrates the expression of the genes necessary for curli fiber assembly (S. Sokaribo et al., 2020). CsgE and CsgF are thought to play important chaperone functions in the assembly of CsgA into curli (Green et al., 2016). CsgG is an oligomeric lipoprotein located in the outer membrane, responsible for stabilizing and exporting the major subunit CsgA and the minor subunit CsgB to the cell surface (Nenninger et al., 2009). CsgA has been shown to bind to small molecular weight proteins mussel foot proteins (Mfps) to maintain their function and be secreted extracellularly (Zhong et al., 2014).
The primary functionality of this construct is to facilitate the expression and secretion of AG4 protein. Successful extracellular secretion of AG4 is indicative of the functional reconstitution of the curli operon and demonstrates the plasmid's capability to support biofilm assembly.
Figure 1 | The function of CsgC and CsgD in JF1 strains. CsgC is involved in preventing the premature polymerization of curli subunits, ensuring that assembly occurs outside the bacterial cell where it's intended. This chaperone-like protein binds to curli subunits and prevents their aggregation inside the cell. CsgD is used as a transcription factor to ensure the expression of the fusion protein CsgA-AG4. 
The specific secretory apparatus are composed of CsgE, CsgF, and CsgG proteins. The amyloid proteins CsgA fold when they secrete through the sepcific channels. If the CsgA proteins do not secrete out, Congo red can not stain them. Congo red only can stain the CsgA proteins which exit the channel successfully. We used Congo red to stain the bacteria solution, which there was no damage to bacteria. The figure shows red sediment which indicates that the CsgA exitted the channel successfully. Therefore, we can prove the functions of the CsgE, CsgF, and CsgG.
Figure 2 | The Congo red staining of bacteria. The red sediment means the bacteria expressed the amyloid. Figure 3 | Congo red staining of polycarbonate membrane with CsgA+Ag4 fusion protein. This picture shows the CsgA-AG4 fusion protein on a polycarbonate membrane (EMD) after simple isolation, purification, and filtration. Here the curli fiber membrane has undergone Congo red staining (amyloid can be stained by Congo red). This could prove that the function of CsgC, and CsgD seem well. Figure 4 |Scanning Electron Microscopy (SEM) analysis. The experimenter used SEM to observe the fusion protein produced by the JF1 strain. The part marked in red in the figure is the CsgA-AG4 fusion protein which can be seen clearly. The magnification of A and B are 5 KX and 20 KX respectively. (done by Yuantest Laboratory).
Furthermore, the ability to adsorb silver ions complements the functionality of AG4, opening up possibilities for applications in metal ion capture and environmental remediation. In order to prove that AG4 silver ion-binding peptide can adsorb silver ions, a model for the adsorption of silver ions by a fusion protein membrane was constructed, and the functionality of the fusion protein membrane was evaluated through four variables: time, temperature, pH, and silver ions, which could confirm that AG4 indeed adsorbed silver ions. This engineered system could prove invaluable in tackling environmental pollution or in the recovery of precious metals from industrial processes.
Figure 5 |Assessment of optimal temperature. This graph indicates the adsorption efficiency of CsgA-AG4 recombinant protein from three strains at various temperatures over an 8-hour period. The optimal temperature is found to be 25℃. Figure 6 |Assessment of optimal time. This graph depicts the adsorption efficiency of CsgA-AG4 recombinant protein from three strains at 25°C across varying adsorption times. The figure highlights the optimal adsorption time is found to be 8 hours. Figure 7 |Assessment of optimal silver ion concentration. This graph illustrates the adsorption efficiency of CsgA-AG4 recombinant protein from three strains at 25℃ across varying silver ion concentrations during an 8-hour incubation period. Figure 8 |The function image of time-temperature-adsorption efficiency. A represents a three-dimensional graph illustrating the adsorption efficiency as a function of temperature and time, obtained through fitting. The face center composite design (α=1) was carried out using the design expert 13. B displays a contour plot of the same function. C and D showcase interactions between temperature and time. The resulting graph is as above. 8 hours and 25℃ are proper for proteins to absorb silver ions.
In summary, the CsgA-AG4-CsgC-CsgD-CsgE-CsgF-CsgG plasmid represents a versatile genetic tool with the potential to restore curli fiber synthesis, express AG4, and serve as a foundation for various applications, including the capture of valuable silver ions. Its design aligns with the need to address the genetic alterations in the JF1 strain and offers opportunities for both basic research and practical environmental solutions.
Source
The sequence of AG4 is from the literature “Solution structure of peptide AG4 used to form silver nanoparticles” (Asn1-Pro-Ser-Ser-Leu-Phe-Arg-Tyr-Leu-Pro-Ser-Asp). The sequence of CsgA, CsgC, CsgD, CsgE, CsgF, and CsgG is from NCBI.
References
Evans, L.M. et al. (2015) ‘The Bacterial Curli System Possesses a Potent and Selective Inhibitor of Amyloid Formation’ Mol Cell, 57(3): 445–455. Available at: https://doi.org/10.1016/j.molcel.2014.12.025
Green, A. et al. (2016) ‘Are the curli proteins CsgE and CsgF intrinsicallydisordered?’ Intrinsically Disord Proteins 4(1): e1130675. Available at: https://doi.org/10.1080/21690707.2015.1130675
Nenninger, A.A., Robinson, L.S. and Hultgren S.J. (2009) ‘Localized and efficient curli nucleatione amyloidrequires the chaperone-likassembly protein CsgF’ BIOLOGICAL SCIENCES 106(3) 900-905. Available at: https://doi.org/10.1073/pnas.0812143106
Seker, U.O.S. and Demir, H.V. (2011) ‘Material Binding Peptides for Nanotechnolgy’ Molecules 16(2),1426-1451. Available at: https://doi.org/10.3390/molecules16021426
Sokaribo, A.S. et al. (2020) ‘Metabolic Activation of CsgD in the Regulation of Salmonella Biofilms’ Microorganisms. 8(7):964. Available at: https://doi:10.3390/microorganisms8070964.
Taylor, J.D. et al. (2016) ‘Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones’ Scientific Reports, bind 6, 24656. Available at: https://doi.org/10.1038/srep24656
Zhong, C. et al. (2014) ‘Strong underwater adhesives made by selfassembling multi-protein nanofibres’ nature nanotechnology 9, 858-866 (2014). Available at: https://doi.org/10.1038/nnano.2014.199
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