Plasmid

Part:BBa_K4875021

Designed by: Cheng Ziyan   Group: iGEM23_Metropolis-Shanghai   (2023-08-25)


Str-KDEL_SBP-α2A-AR-Rluc8


Composite Part: BBa_K4875021 (Str-KDEL_SBP-α2A-AR-Rluc8)

Composite Part: BBa_K4875021 (Str-KDEL_SBP-α2A-AR-Rluc8)

Construction Design

Studies have shown that the 2A adrenergic receptor (2A-AR) in the G-protein-coupled receptor (GPCR) family is associated with Alzheimer’s disease and is a potential target for the treatment of neurodegenerative diseases. Plasmid Str-KDEL_SBP-α2A-AR-Rluc8 is mainly composed of BBa_K4875011, BBa_K4875012, BBa_K4875013, BBa_K4875014, and BBa_K4875019.

Figure 1
Figure 1: Plasmid Map of Str-KDEL_SBP-α2A-AR-Rluc8

Engineering Principle

Our developed RUSH-BRET method detects whether these proteins that can bind to 2A-AR-CT can affect the transport of newly formed 2A-AR (BBa_K4875013) to the cell membrane. This method uses an amino acid fragment (KDEL) residing in the endoplasmic reticulum as a hook protein (BBa_K4875008), which is connected to Streptavidin. In addition, 2A-AR is connected to Streptavidin binding protein (SBP) (BBa_K4875012) and Rluc8 (BBa_K4875014), and the genes of these two components are integrated into a plasmid for co-expression. This way, the newly generated protein is left on the endoplasmic reticulum to stop further transport to the cell membrane. Then, the plasmid was co-expressed with the cell membrane marker protein K-Ras labeled with Venus in one cell. During the experiment, when biotin is added, it binds to Str, disrupting the binding between Str and SBP. SBP-2A-AR Rluc8 is released from the endoplasmic reticulum and transported to the cell membrane. After a period of transportation, SBP-2A-AR Rluc8 reaches the cell membrane, and Rluc8 undergoes bioluminescence resonance energy transfer (BRET) with Venus on Venus K-Ras. The higher the value, the more 2A-AR on the cell membrane.

Figure 2
Figure 2: Schematic diagram of RUSH-BRET assay. Image by Xu Xing.[2]

Experimental Approach

In order to stimulate the transportation of α2A-AR-CT protein to the cell membrane, we used HEK293 cell lines as the host. After the cultivation, we processed cell transfection by PEI to transfer the plasmids, Str KDEL_ SBP-α2A-AR-Ruluc8 and Venus-K-Ras plasmid into HEK 293 cells.

Figure 3
Figure 3: Gel electrophoresis results of colony PCR: Marker-15K; 1,19,20-Negative control; 2-18-Colony PCR samples and 7,10,11 are confirmed positive

The following alignment and base pairs which confirmed that we have obtained the expected plasmid.

Figure 4
Figure 4: Global sequence alignment of sequencing

Characterization/Measurement

BRET Result: Based on the data in table 1 where the value is Venus/Rluc8, we could draw the curve as shown in Figure 5.

table-1
Table1. Fluorescence intensity of RUSH-BRET in different time
Figure 5
Figure 5: The surface expression of α2A-AR was measured by RUSH-based BRET assays

These increasing trends mean that the Str-KDEL_SBP-α2A-AR-Rluc8 structure has been disturbed by the biotin and α2A-AR-Rluc8 move from the endoplasmic reticulum to cell membrane. Rluc8 of SBP-α2AR -Rluc8 replaced the RAS of the Venus-K-RAS on the cell membrane and produced BRET signal that represents the amount of the protein α2A-AR on the membrane.

To conclude, our RUSH-BRET system was working to measure the amount of the protein α2A-AR on the membrane and it could be more reliable for our future research on drug screening. Next move we will design more inquiry experiments to evaluate our RUSH-BRET system and evaluate the candidate proteins we targeted from IP-MS results. Based on our discussion with our instructors, we will test those candidate proteins by building gene knock-out cells to check if they possess the ability to affect the transportation of α2A-AR-CT to the membrane.

In the future, we will try to develop more effective drugs targeted for Alzheimer's disease by targeting the alpha2A-adrenergic receptor in collaboration with pharmaceutical companies.

IP-MS results:

Through the GO data, we can know that our target gene is enriched at the biological process level, with the highest number of genes enriched in translation, and our differential genes are most significantly enriched in this regulation of translation. Through the KEGG data, we can know that the number of genes enriched on Ribosome is the highest, and our differential genes are most significantly enriched in Thermogenesis.

Figure 3
Figure 6: KEGG Pathways for Significantly Expressed Proteins
Figure 4
Figure 7: GOBP for Significantly Expressed Proteins

Based on the IP-MS results in figure 6 and 7, we could further find our candidate proteins that have the potential ability to affect the α-AR-CT transportation to the membrane, and the detailed result could be found in this EXCEL.

IP-MS was completed by Professor Shen's research group at Nanjing University of Traditional Chinese Medicine, providing IP-MS testing service and data analysis service.

References

  1. Boncompain, G., Divoux, S., Gareil, N., De Forges, H., Lescure, A., Latreche, L., Mercanti, V., Jollivet, F., Raposo, G. and Perez, F., 2012. Synchronization of secretory protein traffic in populations of cells. Nature methods, 9(5), pp.493-498.
  2. Xu, X. and Wu, G., 2022. Human C1orf27 protein interacts with α2A-adrenergic receptor and regulates its anterograde transport. Journal of Biological Chemistry, 298(6).
  3. Zhang, F., Gannon, M., Chen, Y., Yan, S., Zhang, S., Feng, W., Tao, J., Sha, B., Liu, Z., Saito, T. and Saido, T., 2020. β-amyloid redirects norepinephrine signaling to activate the pathogenic GSK3β/tau cascade. Science translational medicine, 12(526), p.eaay6931.

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Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 7062
    Illegal SapI site found at 3055


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