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Part contributions |
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Part Number |
Part Name |
Contribution Type I |
Contribution Type |
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GST |
basic part |
New experiment data |
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thrombin site |
basic part |
Literature information |
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α2A-AR-CT |
basic part |
New part |
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Modified core streptavidin |
basic part |
New part |
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KDEL-hook |
basic part |
Literature information |
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IRES |
basic part |
Literature information |
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IL-2- signal sequence |
basic part |
Literature information |
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SBP |
basic part |
Literature information |
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α2A-AR |
basic part |
Literature information |
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SV40 poly(A) signal |
basic part |
Literature information |
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mVenus |
basic part |
Literature information |
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K-Ras |
basic part |
New part |
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HSV TK poly(A) signal |
basic part |
Literature information |
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Str-KDEL_SBP-α2A-AR-Rluc8 |
composite part |
New part |
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Venus-K-Ras |
composite part |
New part |
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GST- α2A-AR-CT |
composite part |
New part |
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The Str-KDEL_SBP-α2A-AR-Rluc8 vector carries CMV enhancer , CMV promoter , T7 promoter, Modified core streptavidin, KDEL-hook, Chimeric intron, IRES, IL-2- signal sequence, SBP, α2A-AR, hRluc, and Str-KDEL_SBP-EGFP-Ecadherin.
Literature Review & Experimental Results:
Str KDEL_ SBP- α2A-AR-Rluc8 is our key research object, and we use Str KDEL_ SBP- α2A-AR-Rluc8 to carry our target protein α2A-AR. Due to the luminescent properties of Venus K-Ras, it is convenient for us to use RUSH BRET to detect the presence of α Content of 2A-AR. The surface expression of α2A-AR was measured by RUSH-based BRET assays shows us that our target protein α. The content of 2A-AR on the cell membrane indicating that our conjecture is successful, α2A-AR will transfer from endoplasmic reticulum to the cell membrane.
BRET Curve of the cell within Part BBa_K4875021
The Venus-K-Ras vector carries CMV enhancer, CMV promoter, SV40 poly(A) signal, mVenus, K-Ras, and HSV TK poly(A) signal.
Literature Review & Experimental Results:
Venus K-Ras plays an important role in RUSH BRET. Due to the fact that the biological component mVenus in Venus K-Ras is a yellow fluorescence protein, Venus K-Ras has fluorescence characteristics. The Rluc8 of α-2A-AR-Rluc8 will bind to mVenus in Venus K-Ras. So Venus K-Ras can make Str KDEL_ SBP- α 2A-AR-Rluc8 has fluorescence characteristics to detect the presence of Number of 2A-ARs.
BBa_K4875000 & BBa_K4875023
The GST- α2A-AR-CT carries GST, tac promoter, thrombin site, α2A-AR-CT, and pGEX-4T-1.
Literature Review & Experimental Results:
Obtained GST- α2A-AR-CT protein through protein purification, which is purified and specifically binds to cell lysate or other proteins, identifies proteins that interact with this protein, and then uses the binding protein as a drug design target to develop therapeutic drugs. This picture is the result of SDS-PAGE which is used to test proteins, P represents precipitate; S represents cell lysate; T represents flow through; W represents wash and E represents elution. GST-α2A-AR-CT protein is found on column E, marker 25KDa, this shows GST-α2A-AR-CT protein is purified from BL21(DE3) bacteria.
SDS-PAGE gel of the protein of BBa_K4875023
We fused a GST tag to the CT terminus of the α2A-AR protein and then purify it to a protein that also contains a GST tag.
Literature Review & Experimental Results:
The GST tag frequently increases the solubility of the fused protein of interest and thus enables its purification and subsequent functional characterization. The GST-tagged protein specifically binds to glutathione immobilized to a matrix (e.g., agarose) and can be easily separated from a cell lysate by a bind-wash-elute procedure. GST-tagged proteins are often used to study protein-protein interactions, again making use of glutathione affinity in a procedure called a GST pull-down assay. The protocol is designed to process 200 ml of E. coli culture expressing intermediate to high amounts of a GST-tagged protein (~25 mg l(-1)).(Schäfer, Frank et al, 2015)
Literature Review & Experimental Results:
Activation of prothrombin is crucial in physiological and pathological coagulation. Various rare diseases involving prothrombin have been described (e.g., hypoprothrombinemia). Anti-prothrombin antibodies in autoimmune disease may be a factor in the formation of the lupus anticoagulant (also known as antiphospholipid syndrome). Hyperprothrombinemia can be caused by the G20210A mutation.
Thrombin, a potent vasoconstrictor and mitogen, is implicated as a major factor in vasospasm following subarachnoid hemorrhage. Blood from a ruptured cerebral aneurysm clots around a cerebral artery, releasing thrombin. This can induce an acute and prolonged narrowing of the blood vessel, potentially resulting in cerebral ischemia and infarction (stroke).
Beyond its key role in the dynamic process of thrombus formation, thrombin has a pronounced pro-inflammatory character, which may influence the onset and progression of atherosclerosis. Acting via its specific cell membrane receptors (protease activated receptors: PAR-1, PAR-3 and PAR-4), which are abundantly expressed in all arterial vessel wall constituents, thrombin has the potential to exert pro-atherogenic actions such as inflammation, leukocyte recruitment into the atherosclerotic plaque, enhanced oxidative stress, migration and proliferation of vascular smooth muscle cells, apoptosis and angiogenesis.(Wikipedia-Thrombin, 2023)
Literature Review & Experimental Results:
Streptavidin is a 52 kDa protein (tetramer) purified from the bacteriumStreptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin(also known as vitamin B7 or vitamin H). With a dissociation constant (Kd) on the order of ≈10−14mol/L, the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature. Streptavidin is used extensively in molecular biology and bionanotechnology due to the streptavidin-biotin complex's resistance to organic solvents, denaturants (e.g. guanidinium chloride), detergents (e.g. SDS, Triton X-100), proteolytic enzymes, and extremes of temperature and pH.(Wikipedia-Streptavidin, 2023)
Literature Review & Experimental Results:
KDEL is a target peptide sequence in mammals and plants located on the C-terminal end of the amino acid structure of a protein. The KDEL sequence prevents a protein from being secreted from the endoplasmic reticulum (ER) and facilitates its return if it is accidentally exported.
A protein with a functional KDEL motif will be retrieved from the Golgi apparatus by retrograde transport to the ER lumen. It also targets proteins from other locations (such as the cytoplasm) to the ER. Proteins can only leave the ER after this sequence has been cleaved off. (Wikipedia-KDEL, 2023)
Literature Review & Experimental Results:
An internal ribosome entry site, abbreviated IRES, is an RNA element that allows for translation initiation in a cap-independent manner, as part of the greater process of protein synthesis. In eukaryotic translation, initiation typically occurs at the 5' end of mRNA molecules, since 5' cap recognition is required for the assembly of the initiation complex. The location for IRES elements is often in the 5'UTR, but can also occur elsewhere in mRNAs. (Internal ribosome entry site-Wikipedia,2023)
Literature Review & Experimental Results:
Spontaneous bacterial peritonitis (SBP) is the development of a bacterial infection in the peritoneum, despite the absence of an obvious source for the infection.It is specifically an infection of the ascitic fluid – an increased volume of peritoneal fluid. Ascites is most commonly a complication of cirrhosis of the liver.It can also occur in patients with nephrotic syndrome. SBP has a high mortality rate.(Ameer MA, et al, 2023)
Literature Review & Experimental Results:
The alpha-2A adrenergic receptor (α2A adrenoceptor), also known as ADRA2A, is an α2 adrenergic receptor, and also denotes the human gene encoding it.(Alpha-2A adrenergic receptor-Wikipedia, 2023)
Literature Review & Experimental Results:
SV40 PolyA (Simian virus 40 PolyA, also called PolyA) sequence is DNA sequence (240 bp) that possesses the activity of transcription termination and can add PolyA tail to mRNA. PolyA contains AATAAA hexanucleotide polyadenylation signal.(Li, S. P. et al, 2012)
This is a coding sequence of Venus-K-Ras.
Literature Review & Experimental Results:
Venus is a basic (constitutively fluorescent) yellow fluorescent protein published in 2006, derived from Aequorea victoria. It is reported to be a rapidly-maturing monomer with moderate acid sensitivity.(Kremers, G. J. et al, 2006)
Due to the luminescent properties of mVenus, as a part of Venus K-Ras, the Venus K-Ras plasmid has luminescent properties. We used its luminescent properties to complete the RUSH-BRET experiment. Detection of cell membrane α2A-AR content by RUSH-BRET. To determine our GST- α2A-AR-CT is a target protein.
This is gene which connected with mVenus.
Literature Review & Experimental Results:
The KRAS gene provides instructions for making a protein called K-Ras that is part of a signaling pathway known as the RAS/MAPK pathway. The protein relays signals from outside the cell to the cell's nucleus. These signals instruct the cell to grow and divide (proliferate) or to mature and take on specialized functions (differentiate). The K-Ras protein is a GTPase, which means it converts a molecule called GTP into another molecule called GDP. In this way the K-Ras protein acts like a switch that is turned on and off by the GTP and GDP molecules. To transmit signals, it must be turned on by attaching (binding) to a molecule of GTP. The K-Ras protein is turned off (inactivated) when it converts the GTP to GDP. When the protein is bound to GDP, it does not relay signals to the cell's nucleus. (MedlinePlus, 2017)
In our experiment, K-Ras connects mVenus and locates on Plasama membrane. It is also an important component of Venus K-Ras, laying the foundation for subsequent target protein content detection.
Literature Review & Experimental Results:
Polyadenylation [poly(A)] signals (PAS) are a defining feature of eukaryotic protein-coding genes. The central sequence motif AAUAAA was identified in the mid-1970s and subsequently shown to require flanking, auxiliary elements for both 3′-end cleavage and polyadenylation of premessenger RNA (pre-mRNA) as well as to promote downstream transcriptional termination. More recent genomic analysis has established the generality of the PAS for eukaryotic mRNA.(Proudfoot N. J., 2011)
GST-tag part: Schäfer, Frank et al. “Purification of GST-Tagged Proteins.” Methods in enzymology vol. 559 (2015): 127-39. doi:10.1016/bs.mie.2014.11.005
Thrombin (2023) Wikipedia. Available at: https://en.wikipedia.org/wiki/Thrombin#Mechanism_of_action (Accessed: 17 September 2023).
Streptavidin (2023) Wikipedia. Available at: https://en.wikipedia.org/wiki/Streptavidin (Accessed: 17 September 2023).
KDEL (amino acid sequence) (2023) Wikipedia. Available at: https://en.wikipedia.org/wiki/KDEL_(amino_acid_sequence) (Accessed: 17 September 2023).
Internal ribosome entry site (2023) Wikipedia. Available at: https://en.wikipedia.org/wiki/Internal_ribosome_entry_site (Accessed: 17 September 2023).
Ameer MA, Foris LA, Mandiga P, et al. Spontaneous Bacterial Peritonitis. [Updated 2023 Aug 8]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK448208/
Alpha-2A adrenergic receptor (2023) Wikipedia. Available at: https://en.wikipedia.org/wiki/Alpha-2A_adrenergic_receptor (Accessed: 17 September 2023).
Li, S. P., Feng, J. J., Wang, H. G., Wang, X. F., & Lv, Z. J. (2012). Yi chuan = Hereditas, 34(1), 113–119. https://doi.org/10.3724/sp.j.1005.2012.00113
Kremers G J , Goedhart J , Munster E B V ,et al.Cyan and Yellow Super Fluorescent Proteins with Improved Brightness, Protein Folding, and FRET Förster Radius,[J].Biochemistry, 2006.DOI:10.1021/bi0516273.
MedlinePlus. Bethesda (MD): National Library of Medicine (US); [updated December 1, 2017]. KRAS gene;Available from: https://medlineplus.gov/genetics/gene/kras/#conditions.
Proudfoot N. J. (2011). Ending the message: poly(A) signals then and now. Genes & development, 25(17), 1770–1782. https://doi.org/10.1101/gad.17268411
