Plasmid Design and Development
Animal Cell Nucleofection
Sensing Device
Sample ORs were selected and analyzed via Western Blotting to determine the size and purity of the antigen protein. The image below is a Western Blot of HT-22 Cells. From left to right: Ladder, Ladder, Control, GPF, OR5P3, OR5A1, OR51E1, OR02C1 OR10G4, and OR2W1. The green bands at ~37 kDa prove that OR proteins were produced after 3 days in culture meaning that primary and secondary antibodies matched and bound to target OR antigens.
HT-22 cells were cultured on an Axion Biosystems Microelectrode Array cell culture dish to measure field potential measurements of depolarization. HT-22 cells nucleofected with OR 2W1 were presented with hexanol, a positive ligand for the OR 2W1 receptor. Multiple additions were made in 1 minute increments revealing the resetting capability and max depolarization of the HT-22 cells.
Plasmid design and development is a key component of the eNose project. Since odorant receptor protein function was tested in a mammalian cell line, the plasmids used to drive expression of these proteins were specifically designed for mammalian cells. For example, the odorant receptor proteins OR10G4 and OR2W1 were expressed using the pCMV6 and pCI plasmids, respectively, and both contain a cytomegalovirus (CMV) promoter region for high gene expression levels in mammalian cells.
OR transfected cells were also captured, displayed and analyzed via fluorescence and confocal microscopy. Primary immunohistochemistry exploits a target OR antigen, while secondary immunohistochemistry proves the initial attachment of the primary antibody. The images below show both fluorescence (top) and confocal (bottom) microscopy.
Two prototypes have been designed and developed capable of utilizing different methods of biosensor detection. One of the devices utilizes a photo sensor connected to an Arduino, translating the cell action potentials into plain text.
RESULTS
Demonstration
My New Channel
Here we present results from three different components: 1) Plasmid design and development, 2) Animal Cell Nucleofection, and 3) Device design.
Shown are figures of the PCR that amplified the coding sequences for OR2W1 and OR10G4, and images of gels for the restriction enzyme digests confirming the production of the OR10G4 in pSB1C3 and OR2W1 in pSB1C3 deliverables.
For the PCR reactions, the expected bands for the OR10G4 and OR2W1 are both about 0.95kb (above).
For the OR10G4 restriction enzyme digest reactions, the expected products are (right):
2.3kb and 0.7kb for the EcoRI and BamHI reaction
2.3kb and 0.7kb for the BlpI and PstI reaction
For the OR2W1 restriction enzyme digest reactions, the expected products are (left):
2.2kb and 0.8kb for the BglII and PstI reaction
1.8kb and 1.2kb for the MscI reaction.
Additionally, since olfactory receptors are from the same family of proteins, other sequences were examined under restriction enzyme digest but not inserted into the pSB1C3 plasmid (below).
Transferring the olfactory receptor into animal cells is critical component to the development of a working biosensor. However, determining the production of the olfactory receptor was determined by three methods: 1) Western Blotting to determine if the olfactory receptor protein was produced, 2) immunohistochemistry ensure the receptor was transported to the membrane, and 3) physiological response of cell action potentials in the presence of specific odorants.
In a controlled environment, neurons with enhanced olfactory receptors are cultured on a microelectrode array. While media is flowed through via a peristaltic pump, certain olfactants are injected. When the neurons detect these olfactants, they produce an electrical signal, which is then transferred to a computer for interpretation.
After conducting multiple trials, it was shown that there was no significant increase in voltage reading the olfactant was introduced into the closed system. This may have been because the plasmid is not being expressed sufficiently, or that 24 hours after transfection was not the optimal time to conduct trials. There was also no reference electrode, which could have altered the computers interpretation of the voltage data.
Western Blot
Immunohistochemistry
Physiological Response
Caption: Confocal microscopy maximum intensity projection images of 6 transfected ORs showing specific and non-specific binding.
Caption: Electrophysiology of HT-22 cells on Axion Biosystems microelectrode array (MEA).
Microelectrode Array Bioreactor
Handheld Photosensor Device
For construction of iGEM deliverables containing odorant receptor proteins in the pSB1C3 plasmid, the coding sequences for OR10G4 and OR2W1 were amplified by PCR using the respective pCMV6 and pCI mammalian expression plasmids as templates. The PCR reactions were expected to generate bands of approximately 0.95kb for both OR10G4 and OR2W1 coding sequences, and the image of the gel for the PCR products shows bands at this expected size. These PCR products were amplified using primers that incorporated EcoRI and PstI restriction endonuclease sites at the 5’ and 3’ ends of the PCR products. Digesting both the PCR products and the pre-linearized pSB1C3 plasmid supplied by iGEM with EcoRI and PstI restriction endonucleases, the PCR products were ligated into the pSB1C3 plasmid. The ligation reaction was used to transform chemically competent DH5alpha cells. To screen for colonies containing the appropriately inserted odorant receptor gene and pSB1C3 plasmid, colonies were used to inoculate small cultures. The plasmids were harvested from the cultures by mini-prep plasmid isolation kits, and the isolated plasmids were digested with two sets of restriction endonuclease reactions.