Part:BBa_K5304007
Plac(lacO)::Lpp'::OmpA(46-159)-RGD
The Plac(lacO)::Lpp'::OmpA(46-159)-RGD part drives constitutive expression of the Lpp'-OmpA-RGD(46-159) fusion protein, which displays the RGD peptide on the bacterial outer membrane via OmpA, facilitating targeted binding to tumor cells by interacting with integrins.
Background
Surface display systems are powerful tools in synthetic biology, allowing for the presentation of functional peptides or proteins on the surface of bacteria. The Lpp'-OmpA chimaera is one such surface display system, widely used for anchoring proteins to the bacterial outer membrane. RGD peptides have a well-established role in targeting tumors due to their affinity for integrins, such as αvβ3, which are overexpressed in many types of tumors. In this part, Lpp' directs the fusion protein to the bacterial outer membrane, while OmpA provides transmembrane domains that enable efficient surface display of the RGD peptide.
Usage and Biology
The plac(ΔlacO)::Lpp’::OmpA(46-159)-RGD is designed to enhance the targeting of tumor cells by Salmonella. The plac(ΔlacO) promoter, which was modified to be constitutive by removing the lac operator, drives the continuous expression of the fusion protein. The Lpp' signal directs the fusion protein to the bacterial outer membrane, while OmpA provides the necessary transmembrane regions for proper surface display of the RGD peptide. The RGD peptide binds integrins, such as αvβ3, that are commonly overexpressed on the surface of tumor cells, allowing Salmonella to specifically target and deliver therapeutic payloads to the tumor microenvironment. It can be used in bacterial tumor therapies or in any application where selective targeting of integrins is desired.
Design
The design of the plac(ΔlacO)::Lpp’::OmpA(46-159)-RGD part focuses on achieving continuous expression of the fusion protein and its efficient display on the bacterial surface. The plac(ΔlacO) promoter was modified for constitutive expression, and the Lpp'-OmpA structure was chosen for reliable outer membrane anchoring and surface presentation of the RGD peptide, which enhances tumor targeting through integrin binding. This modular design allows for flexibility in future modifications or applications. The whole sequence was incorporated into the plasmid pSilencer-CLDN6 to facilitate functional testing and expression in the bacterial system.
Figure 1. plasmid pSilencer-CLDN6 Drugs.
Validation of RGD Expressiony
To validate the surface expression of the RGD peptide on Salmonella, flow cytometry analysis was conducted. Two groups were analyzed: the control group (Salmonella strain χ11803 without the plasmid) and the experimental group (Salmonella strain χ11803 carrying the pSilencer-CLDN6 plasmid with the plac(ΔlacO)::Lpp’::OmpA(46-159)-RGD construct.
Figure 2. Validation of RGD expression using flow cytometry.
The flow cytometry result, as shown in the graph, indicates a significant shift in fluorescence intensity between the control and experimental groups. The control group, represented by the red curve, shows a low fluorescence signal, confirming that the RGD peptide is not expressed in the absence of the plasmid. In contrast, the experimental group, represented by the blue curve, shows a clear shift towards higher fluorescence intensity, indicating successful expression of the RGD peptide on the surface of the bacteria. The result validates that the plac(ΔlacO)::Lpp’::OmpA(46-159)-RGD construct was effectively expressed, and the RGD peptide was displayed on the outer membrane of Salmonella, confirming the functional design of the system.
Conclusion and Outlook
In conclusion, the plac(ΔlacO)::Lpp’::OmpA(46-159)-RGD composite part successfully facilitated the surface display of the RGD peptide on Salmonella, as validated by flow cytometry. This system demonstrates the feasibility of using Salmonella as a platform for tumor-targeting bacterial therapies. The inclusion of the RGD peptide, which binds integrins overexpressed on tumor cells, enhances the specificity of bacterial targeting and improves the therapeutic potential. Looking forward, this surface display system offers significant flexibility. By replacing the RGD peptide with other targeting molecules, such as antibodies or specific peptides, the system can be adapted to suit a variety of therapeutic purposes. This versatility allows the development of bacterial therapies aimed at different diseases or targeting different cell types, extending its application. This part contributes to the iGEM community by providing a modular and adaptable tool for engineering bacteria. The potential for customized targeting could lead to more precise therapeutic delivery systems. In terms of societal impact, this system holds promise for advancing personalized medicine, offering safer and more effective treatment options with reduced side effects.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |