Introduction
Our project focuses on developing a novel approach by engineering exosomes to deliver essential proteins to cancer cells, stimulating calcium overloading and inducing cancer cell death. This strategy involves introducing calcium ion channels (MscS or hTRPC1) and an NO signaling activator (hNOS2) using exosomes as the delivery vehicle.
Basic Parts
MscS
Part Code
BBa_K5353001
Part Type
Coding
Description
MscS’s function involves facilitating turgor regulation in bacteria and responding to alterations in osmotic pressure (Perozo, 2003). Previous studies have demonstrated that MscS forms a homoheptameric structure, with each subunit weighing 31kDa and comprising three transmembrane helices. The N-terminus is oriented towards the periplasm, while the C-terminus is integrated into the cytoplasm (Pivetti, 2003).
By introducing the MscS calcium channels into cancer cells, we aim to generate calcium overload. Ultrasound stimulation will precisely control cell death by triggering the opening of channels and leading to calcium ion influx.
hTRPC1
Part Code
BBa_K5353002
Part Type
Coding
Description
hTRPC1, a transient receptor potential channel in humans, is a non-specific cation channel positioned on the cell's plasma membrane. Studies have indicated its widespread presence across human tissues, where it operates as a calcium channel activated by stored calcium (Xu, 2001). It belongs to the TRP superfamily. These tetrameric transmembrane proteins are structured with six helices creating a pore between S5 and S6 (Nilius, 2007). These helices orient the N- and C-termini towards the cell's interior, thereby facilitating the assembly of functional homo- or hetero-tetramers (Bianchi, 2007).
By introducing the hTRPC1 calcium channels into cancer cells, we aim to generate calcium overload. Ultrasound stimulation will precisely control cell death by triggering the opening of channels and leading to calcium ion influx.
hNOS2
Part Code
BBa_K5353003
Part Type
Coding
Description
The hNOS2 plays a crucial role in the immune response by catalyzing the production of nitric oxide (NO) from L-arginine. Unlike constitutively expressed NOS isoforms, NOS2 is typically induced in response to inflammatory stimuli such as cytokines, pathogens, and endotoxins.
The hNOS2 induces NO signaling by catalyzing the reaction: L-arginine + O2 + NADPH + H+ → NO + L-citrulline + NADP+ + H2O. Upregulation of the NO signal can induce the opening of the RyR calcium channel on the endoplasmic reticulum, dramatically increasing intracellular calcium levels (Ziolo et al., 2001).
Lamp2b
Part Code
BBa_K5353000
Part Type
Coding
Description
Lamp2b is primarily found on the membrane of lysosomes, which are essential for the degradation and recycling of cellular components, and it is involved in the fusion of lysosomes with autophagosomes during the process of autophagy, where cellular components are degraded to maintain cellular homeostasis.
Lamp2b has been widely used in the manufacture of engineered exosomes for targeted drug delivery (Qiao et al., 2023). And we utilize lamp2b, fused with our target proteins, to enhance the loading of MscS, hTRPC1, and hNOS2 into the exosomes. With exosomes as our delivery vehicle, we aim to deliver our target proteins to cancer cells to induce the cancer cell calcium overload.
EGFP
Part Code
BBa_K5353011
Part Type
Reporter
Description
BBa_K5353011 is a modified version of the Green Fluorescent Protein (GFP), with brighter fluorescence that makes the fluorescence microscopey, fluorometertry, fluorescence-activated cell sorting (FACS), or imaging in a microplate reader more sensitive. It can act as a marker in HEK293T cells to ondicate sucessufully infection and facilitate selection using fluorescence-activated cell sorting (FACS), for collection of cells for exosome extration.
mCherry
Part Code
BBa_K5353012
Part Type
Reporter
Description
mCherry is a part of the mFruits protein family, which is a family of mRFPs, monomeric red fluorescent proteins. With fluorescence microscope detection, we can identify which HEK293T cell was sucessufully infected and select them with fluorescence-activated cell sorting (FACS) followed by collection for exosome extraction.
HA
Part Code
BBa_K5353021
Part Type
Tag
Description
The HA (hemagglutinin) tag is derived from the human influenza virus HA protein. We link the tag to MscS and hTRPC1 for further detection. HA tag antibodies provide a robust method for the detection of tagged target proteins without need for a protein-specific antibody or probe.
Flag
Part Code
BBa_K5353022
Part Type
Tag
Description
Flag-tag is a commonly used epitope or peptide tag that is not derived from a natural protein. It is used to tag proteins for multiple capture and detection applications. The hydrophilic protein tag is commonly used in conjunction with antibodies in protein pull-downs to study protein–protein interactions. We link the tag to the hNOS2 for further detection.
EF1A
Part Code
BBa_K5353031
Part Type
Promoter
Description
The EF1A promoter, derived from the human elongation factor 1 alpha (EF1A) gene initiates the transcription of a downstream gene by providing the binding site for RNA polymerase and transcription factors. The EF1A promoter drives high levels of gene expression in a stable and consistent manner, making it suitable for target protein expression.
CMV
Part Code
BBa_K5353032
Part Type
Promoter
Description
The Cytomegalovirus (CMV) promoter is a strong and commonly used promoter sequence derived from the immediate early gene of human cytomegalovirus. It is widely used for driving gene expression in mammalian cells while it initiates the transcription of a downstream gene by providing the binding site for RNA polymerase and transcription factors. Therefore, it is suitable for our target protein expression by the HEK293T cell.
Composite Parts
LentiV-MscS/HA: EGFP/Neo
Part Code
BBa_K5353061
Part Type
Plasmid
Description
After purifying the plasmid DNA through midi-prep, we utilize them to package the lentivirus together with DVPR and VSV-G. We utilize these lentivirus to infect HEK293T cell and the successfully infected cells will express the MscS-HA tag protein. Meanwhile, this plasmid will also express green fluorescent protein (GFP). The MscS could be induced by physical stimulation (such as ultrasound) to induce the calcium overload. The HA tag could be used for Western blot and imaging detection of the MscS protein, while GFP can be used for validation of the lentivirus infection. The lentivirus gene sequence could be integrated into HEK293T cell genome by utilizing G418 selection (by the Neo marker gene).
Component Parts
BBa_K5353001, BBa_K5353021, BBa_K5353011, BBa_K5353031, BBa_K5353032
LentiV-Lamp2b/MscS/HA: EGFP/Neo
Part Code
BBa_K5353062
Part Type
Plasmid
Description
After purifying the plasmid DNA through midi-prep, we utilize them to package the lentivirus together with DVPR and VSV-G. We utilize these lentivirus to infect HEK293T cell and the successfully infected cells will express a fusion protein that combines Lamp2b, MscS, and HA tag, the plasmid will also express green fluorescent protein (GFP). The Lamp2b sequence is for the transportation of MscS to the exosome. The MscS is the ion channel for the calcium overloading in cancer cells. The HA tag will be used for the detection of the target protein, while GFP can be used to validate infection rate. The lentivirus gene sequence could be integrated into the HEK293T cell genome by utilizing G418 selection (by the Neo gene).
Component Parts
BBa_K5353000, BBa_K5353001, BBa_K5353021, BBa_K5353011, BBa_K5353031, BBa_K5353032
LentiV-Lamp2b/MscS/mCherry: Neo
Part Code
BBa_K5353063
Part Type
Plasmid
Description
After purifying the plasmid DNA through midi-prep, we utilize them to package the lentivirus together with DVPR and VSV-G. We utilize these lentivirus to infect HEK293T cell and the successfully infected cells will express a fusion protein: Lamp2b-MscS-mCherry. The lentivirus gene sequence could be integrated into the HEK293T cell genome by utilizing G418 selection (by the Neo gene).
Component Parts
LentiV-hTRPC1/HA: EGFP/Neo
Part Code
BBa_K5353064
Part Type
Plasmid
Description
After purifying the plasmid DNA through midi-prep, we utilize them to package the lentivirus together with DVPR and VSV-G. We utilize these lentivirus to infect HEK293T cell and the successfully infected cells will express hTRPC1 with HA-Tag. hTRPC1 is a cation channel that is permeable to calcium (Ca2+) and will be used for the calcium overload. The lentivirus gene sequence could be integrated into the HEK293T cell genome by utilizing by utilizing G418 selection (by the Neo gene).
Component Parts
BBa_K5353002, BBa_K5353021, BBa_K5353011, BBa_K5353031, BBa_K5353032
LentiV-Lamp2b/hTRPC1/HA: Neo
Part Code
BBa_K5353065
Part Type
Plasmid
Description
After purifying the plasmid DNA through midi-prep, we utilize them to package the lentivirus together with DVPR and VSV-G. We utilize these lentivirus to infect HEK293T cell and the successfully infected cells will express the fusion protein of Lamp2b-hTRPC1 with HA-Tag. The Lamp2b sequence is for the transportation of hTRPC1 to the exosome. hTRPC1 is a cation channel that is permeable to calcium (Ca2+) and will be used for the calcium overload. The lentivirus gene sequence could be integrated into the HEK293T cell genome by utilizing G418 selection (by the Neo gene).
Component Parts
BBa_K5353000, BBa_K5353002, BBa_K5353021, BBa_K5353032
LentiV-hNOS2/Flag: Puro
Part Code
BBa_K5353066
Part Type
Plasmid
Description
After purifying the plasmid DNA through midi-prep, we utilize them to package the lentivirus together with DVPR and VSV-G. We utilize these lentivirus to infect HEK293T cells and the successfully infected cells will express the hNOS2 gene with Flag-tag. The hNOS will be used to trigger the release of the intracellular calcium. The Flag-tag will be used for detection. The lentivirus gene sequence could be integrated into the HEK293T cell genome by utilizing Puromycin (by the Puro resistance gene).
Component Parts
BBa_K5353003, BBa_K5353022, BBa_K5353031
