Part:BBa_K5283027
P32 promoter-SPusp45-LEISSTCDA-acmA-Linker-P9-FLAG
Compared to plasmid BBa_K5283026, we have additionally incorporated the FLAG tag, a commonly used tag sequence for the detection and purification of proteins, which aids in tracking and verifying protein expression in experiments.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 221
Illegal PstI site found at 955
Illegal PstI site found at 2284
Illegal PstI site found at 2656 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 221
Illegal PstI site found at 955
Illegal PstI site found at 2284
Illegal PstI site found at 2656 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1035
Illegal BamHI site found at 1395
Illegal BamHI site found at 2889 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 221
Illegal PstI site found at 955
Illegal PstI site found at 2284
Illegal PstI site found at 2656 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 221
Illegal PstI site found at 955
Illegal PstI site found at 2284
Illegal PstI site found at 2656 - 1000COMPATIBLE WITH RFC[1000]
Plasmid Construction
We chose pMG36e as the plasmid scaffold, modified into a constitutive promoter p32.
Most diabetes patients are also accompanied by disruption of islet beta cells, a decrease in baseline insulin levels in the later stages of the disease, which leads to an increase in daily blood glucose levels. Therefore, we chose P9 to control the blood glucose of patients by raising the baseline insulin level. The P9 molecule binds to the ICAM-2 receptor on the intestinal secretory L cell in the intestinal crypt, thereby efficiently inducing the intestinal gland to gently release GLP-1 into the bloodstream. Due to the limited number of secreted proteins of lactic acid bacteria, and the relatively long time for GLP-1 to play its role after reaching the islets through the blood, we chose to use *P32* component promoter (BBa_K5283015) to express P9 on the surface of L. lactis strain NZ9000 via cA-anchor(BBa_K5283020). In this way, excessive consumption of intracellular resources of can be avoided. After culture in vitro under high nutrient conditions for a period of time, P9 on its surface can reach a relatively high level.
Modeling and Simulation
Protein Stability Prediction Regarding the P9 protein, there is currently no research on its active sites. Creating fusion proteins of P9-cA.Anchor with novel structures and functions implies the need for protein design. De novo protein design is a long-standing fundamental goal of synthetic biology, but this complex and challenging task is primarily hindered by the difficulty of reliably predicting the 3D structure of proteins from amino acid sequences. Machine learning algorithms, such as AlphaFold2, may eliminate this obstacle. Therefore, we utilized AlphaFold2 for molecular docking (Zhenyu Yang et al., 2003). In Figure 3, the green portion represents the P9 protein, the yellow represents the P9 protein receptor ICAM2, and the light blue is the cAAnchor protein. The outcomes of the molecular docking demonstrate that the key interaction sites are situated at asparagine at position 633 to valine at position 636.
Hydropathy predictions suggest that the P9 protein and the cA.Anchor protein exhibit robust hydrophobic interactions, which promote their tight binding in aqueous solutions instead of forming a loose structure that might potentially conceal active sites.
Expression and Verification
We initially incorporated a FLAG-tag into the P9 fusion protein for subsequent identification. By utilizing antibodies that specifically bind to FLAG-tag and emit fluorescence, we conducted staining on the induced bacteria and observed afterwards. The engineered bacteria demonstrated green fluorescence emitted by the antibodies on the surface of the cells, while the wild-type bacteria performed no fluorescence, indicating that the P9 fusion protein was successfully anchored to the bacteria surface.
Function Verification
To test the effectiveness of the biological function of P9 (BBa_K5283019) expressed on the surface of our engineered L. lactis strain NZ9000, NCI-H716 cells were co-cultured with the engineered L. lactis strain NZ9000 for 2 h. After that, the cell supernatant was collected for GLP-1 concentration measurement, and the NCI-H716 cells were used for RT-PCR to assess the expression levels of Gcg, Pcsk1 and Pcsk2. We treated the NCI-H716 cells with four different conditions: no engineered L. lactis strain NZ9000 added, IPTG+, IPTG-, and IPTG+ with trypsin-EDTA.
By measuring the GLP-1 concentration through ELISA, we found that the GLP-1 secretion level in the IPTG+ group was significantly higher than that in the other treatment groups.
The transcription levels of Gcg,Pcsk1 and Pcsk2 were measured by RT-PCR for each group of cells. Compared to the other treatment groups, the expression of Gcg and Pcsk1 in the IPTG+ group was more pronounced.
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