iGEM 2017 — Team USMA-West_Point

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# Team:USMA-West Point/Notebook

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[DELIVERABLES](https://2017.igem.org/Team%3AUSMA-West_Point/Deliverables)

PARTS

[Parts](https://2017.igem.org/Team%3AUSMA-West_Point/Parts)
[Basic Parts](https://2017.igem.org/Team%3AUSMA-West_Point/Basic_Part)
[Part Collection](https://2017.igem.org/Team%3AUSMA-West_Point/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3AUSMA-West_Point/Safety)

HUMAN PRACTICES

[Integrated and Silver/Gold](https://2017.igem.org/Team%3AUSMA-West_Point/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3AUSMA-West_Point/Engagement)

AWARDS

[Entrepreneurship](https://2017.igem.org/Team%3AUSMA-West_Point/Entrepreneurship)
[Applied Design](https://2017.igem.org/Team%3AUSMA-West_Point/Applied_Design)
[Hardware](https://2017.igem.org/Team%3AUSMA-West_Point/Hardware)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=USMA-West_Point)

# USMA-West\_Point

Notebook - iGem 01

[## usMA-WEST POINT](https://2017.igem.org/Team%3AUSMA-West_Point)

* [Home](https://2017.igem.org/Team%3AUSMA-West_Point)
* [Team](/free/amitro02/igem-01/vbid-f02482b7-t3dtwkay)
* [Students, Advisors, PIs, and Instructors](https://2017.igem.org/Team%3AUSMA-West_Point/Team)
* [Collaboration](https://2017.igem.org/Team%3AUSMA-West_Point/Collaborations)

- Project
- [Project](https://2017.igem.org/Team%3AUSMA-West_Point/Project)
- [Description](https://2017.igem.org/Team%3AUSMA-West_Point/Description)
- [Design](https://2017.igem.org/Team%3AUSMA-West_Point/Design)
- [Experiments](https://2017.igem.org/Team%3AUSMA-West_Point/Experiments)
- [Notebook](https://2017.igem.org/Team%3AUSMA-West_Point/Notebook)
- [InterLab Study](https://2017.igem.org/Team%3AUSMA-West_Point/InterLab)
- [Results](https://2017.igem.org/Team%3AUSMA-West_Point/Results)
- [Demonstrate](https://2017.igem.org/Team%3AUSMA-West_Point/Demonstrate)
- [Attribution](https://2017.igem.org/Team%3AUSMA-West_Point/Attributions)

- [Deliverables](https://2017.igem.org/Team%3AUSMA-West_Point/Deliverables)
- Parts
- [Parts](https://2017.igem.org/Team%3AUSMA-West_Point/Parts)
- [Basic Parts](https://2017.igem.org/Team%3AUSMA-West_Point/Basic_Part)
- [Parts Collection](/free/amitro02/igem-01/https%3A//2017.igem.org/Team%3AUSMA-West_Point/Part_Collection)

- [Safety](https://2017.igem.org/Team%3AUSMA-West_Point/Safety)
- Human Practices
- [Integrated and Gold](https://2017.igem.org/Team%3AUSMA-West_Point/HP/Gold_Integrated)
- [Public Engagement](https://2017.igem.org/Team%3AUSMA-West_Point/Engagement)

- Awards
- [Entreneurship](https://2017.igem.org/Team%3AUSMA-West_Point/Entrepreneurship)
- [Applied design](https://2017.igem.org/Team%3AUSMA-West_Point/Applied_Design)
- [Hardware](https://2017.igem.org/Team%3AUSMA-West_Point/Hardware)

# NOTEBOOK

## January 2017

![](https://lh3.googleusercontent.com/EWqW7DEI4kOTRMLjK2-ObFHp-EYBt5apFYZ1LVFAhLtTLjigCRfx5hCCTKbIjIm68VQ00p9twloHJ9w8=s50)
![](https://lh3.googleusercontent.com/TgRyMQvJ3_h9RmOnu7AlhIE7NLOOBsRoBounARrs8fQv8HCRPaFtpBneSqJOSZpI6l7He_bAZKN179JBig=s50)
![](https://lh3.googleusercontent.com/43-pXHjwrpmVO8Oean-6BD0uzARvcqUQrpdi7Yw2bxaXwEoP21UdN5kW6Ks9pdOxf7ropMUrh0djgYPwYPU=s50)
![](https://lh3.googleusercontent.com/9rwgVnDglPdPFugSu98fhDmxzjXC9KovZ_7BuHkXPIv6jvg9S96flGnhL_e4y8mIpPpZQstfqEV-WitY=s50)

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# Team:USMA-West Point/Experiments

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[Project](https://2017.igem.org/Team%3AUSMA-West_Point/Project)
[Description](https://2017.igem.org/Team%3AUSMA-West_Point/Description)
[Design](https://2017.igem.org/Team%3AUSMA-West_Point/Design)
[Experiments](https://2017.igem.org/Team%3AUSMA-West_Point/Experiments)
[Notebook](https://2017.igem.org/Team%3AUSMA-West_Point/Notebook)
[InterLab](https://2017.igem.org/Team%3AUSMA-West_Point/InterLab)
[Results](https://2017.igem.org/Team%3AUSMA-West_Point/Results)
[Demonstrate](https://2017.igem.org/Team%3AUSMA-West_Point/Demonstrate)
[Attributions](https://2017.igem.org/Team%3AUSMA-West_Point/Attributions)

[DELIVERABLES](https://2017.igem.org/Team%3AUSMA-West_Point/Deliverables)

PARTS

[Parts](https://2017.igem.org/Team%3AUSMA-West_Point/Parts)
[Basic Parts](https://2017.igem.org/Team%3AUSMA-West_Point/Basic_Part)
[Part Collection](https://2017.igem.org/Team%3AUSMA-West_Point/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3AUSMA-West_Point/Safety)

HUMAN PRACTICES

[Integrated and Silver/Gold](https://2017.igem.org/Team%3AUSMA-West_Point/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3AUSMA-West_Point/Engagement)

AWARDS

[Entrepreneurship](https://2017.igem.org/Team%3AUSMA-West_Point/Entrepreneurship)
[Applied Design](https://2017.igem.org/Team%3AUSMA-West_Point/Applied_Design)
[Hardware](https://2017.igem.org/Team%3AUSMA-West_Point/Hardware)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=USMA-West_Point)

# USMA-West\_Point

Experiments - iGem 01

[## usMA-WEST POINT](https://2017.igem.org/Team%3AUSMA-West_Point)

* [Home](https://2017.igem.org/Team%3AUSMA-West_Point)
* [Team](/free/amitro02/igem-01/vbid-f02482b7-t3dtwkay)
* [Students, Advisors, PIs, and Instructors](https://2017.igem.org/Team%3AUSMA-West_Point/Team)
* [Collaboration](https://2017.igem.org/Team%3AUSMA-West_Point/Collaborations)

- Project
- [Project](https://2017.igem.org/Team%3AUSMA-West_Point/Project)
- [Description](https://2017.igem.org/Team%3AUSMA-West_Point/Description)
- [Design](https://2017.igem.org/Team%3AUSMA-West_Point/Design)
- [Experiments](https://2017.igem.org/Team%3AUSMA-West_Point/Experiments)
- [Notebook](https://2017.igem.org/Team%3AUSMA-West_Point/Notebook)
- [InterLab Study](https://2017.igem.org/Team%3AUSMA-West_Point/InterLab)
- [Results](https://2017.igem.org/Team%3AUSMA-West_Point/Results)
- [Demonstrate](https://2017.igem.org/Team%3AUSMA-West_Point/Demonstrate)
- [Attribution](https://2017.igem.org/Team%3AUSMA-West_Point/Attributions)

- [Deliverables](https://2017.igem.org/Team%3AUSMA-West_Point/Deliverables)
- Parts
- [Parts](https://2017.igem.org/Team%3AUSMA-West_Point/Parts)
- [Basic Parts](https://2017.igem.org/Team%3AUSMA-West_Point/Basic_Part)
- [Parts Collection](https://2017.igem.org/Team%3AUSMA-West_Point/Part_Collection)

- [Safety](https://2017.igem.org/Team%3AUSMA-West_Point/Safety)
- Human Practices
- [Integrated and Gold](https://2017.igem.org/Team%3AUSMA-West_Point/HP/Gold_Integrated)
- [Public Engagement](https://2017.igem.org/Team%3AUSMA-West_Point/Engagement)

- Awards
- [Entreneurship](https://2017.igem.org/Team%3AUSMA-West_Point/Entrepreneurship)
- [Applied design](https://2017.igem.org/Team%3AUSMA-West_Point/Applied_Design)
- [Hardware](https://2017.igem.org/Team%3AUSMA-West_Point/Hardware)

# Experiments

## Methods

![](https://lh3.googleusercontent.com/EWqW7DEI4kOTRMLjK2-ObFHp-EYBt5apFYZ1LVFAhLtTLjigCRfx5hCCTKbIjIm68VQ00p9twloHJ9w8=s50)
![](https://lh3.googleusercontent.com/TgRyMQvJ3_h9RmOnu7AlhIE7NLOOBsRoBounARrs8fQv8HCRPaFtpBneSqJOSZpI6l7He_bAZKN179JBig=s50)
![](https://lh3.googleusercontent.com/43-pXHjwrpmVO8Oean-6BD0uzARvcqUQrpdi7Yw2bxaXwEoP21UdN5kW6Ks9pdOxf7ropMUrh0djgYPwYPU=s50)
![](https://lh3.googleusercontent.com/9rwgVnDglPdPFugSu98fhDmxzjXC9KovZ_7BuHkXPIv6jvg9S96flGnhL_e4y8mIpPpZQstfqEV-WitY=s50)

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# Team:USMA-West Point/Results

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MENU

[HOME](https://2017.igem.org/Team%3AUSMA-West_Point)

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[Team](https://2017.igem.org/Team%3AUSMA-West_Point/Team)
[Collaborations](https://2017.igem.org/Team%3AUSMA-West_Point/Collaborations)

PROJECT

[Project](https://2017.igem.org/Team%3AUSMA-West_Point/Project)
[Description](https://2017.igem.org/Team%3AUSMA-West_Point/Description)
[Design](https://2017.igem.org/Team%3AUSMA-West_Point/Design)
[Experiments](https://2017.igem.org/Team%3AUSMA-West_Point/Experiments)
[Notebook](https://2017.igem.org/Team%3AUSMA-West_Point/Notebook)
[InterLab](https://2017.igem.org/Team%3AUSMA-West_Point/InterLab)
[Results](https://2017.igem.org/Team%3AUSMA-West_Point/Results)
[Demonstrate](https://2017.igem.org/Team%3AUSMA-West_Point/Demonstrate)
[Attributions](https://2017.igem.org/Team%3AUSMA-West_Point/Attributions)

[DELIVERABLES](https://2017.igem.org/Team%3AUSMA-West_Point/Deliverables)

PARTS

[Parts](https://2017.igem.org/Team%3AUSMA-West_Point/Parts)
[Basic Parts](https://2017.igem.org/Team%3AUSMA-West_Point/Basic_Part)
[Part Collection](https://2017.igem.org/Team%3AUSMA-West_Point/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3AUSMA-West_Point/Safety)

HUMAN PRACTICES

[Integrated and Silver/Gold](https://2017.igem.org/Team%3AUSMA-West_Point/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3AUSMA-West_Point/Engagement)

AWARDS

[Entrepreneurship](https://2017.igem.org/Team%3AUSMA-West_Point/Entrepreneurship)
[Applied Design](https://2017.igem.org/Team%3AUSMA-West_Point/Applied_Design)
[Hardware](https://2017.igem.org/Team%3AUSMA-West_Point/Hardware)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=USMA-West_Point)

# USMA-West\_Point

Results - iGem 01

[## usMA-WEST POINT](https://2017.igem.org/Team%3AUSMA-West_Point)

* [Home](https://2017.igem.org/Team%3AUSMA-West_Point)
* [Team](/free/amitro02/igem-01/vbid-f02482b7-t3dtwkay)
* [Students, Advisors, PIs, and Instructors](https://2017.igem.org/Team%3AUSMA-West_Point/Team)
* [Collaboration](https://2017.igem.org/Team%3AUSMA-West_Point/Collaborations)

- Project
- [Project](https://2017.igem.org/Team%3AUSMA-West_Point/Project)
- [Description](https://2017.igem.org/Team%3AUSMA-West_Point/Description)
- [Design](https://2017.igem.org/Team%3AUSMA-West_Point/Design)
- [Experiments](https://2017.igem.org/Team%3AUSMA-West_Point/Experiments)
- [Notebook](https://2017.igem.org/Team%3AUSMA-West_Point/Notebook)
- [InterLab Study](https://2017.igem.org/Team%3AUSMA-West_Point/InterLab)
- [Results](https://2017.igem.org/Team%3AUSMA-West_Point/Results)
- [Demonstrate](https://2017.igem.org/Team%3AUSMA-West_Point/Demonstrate)
- [Attribution](https://2017.igem.org/Team%3AUSMA-West_Point/Attributions)

- [Deliverables](https://2017.igem.org/Team%3AUSMA-West_Point/Deliverables)
- Parts
- [Parts](https://2017.igem.org/Team%3AUSMA-West_Point/Parts)
- [Basic Parts](https://2017.igem.org/Team%3AUSMA-West_Point/Basic_Part)
- [Parts Collection](https://2017.igem.org/Team%3AUSMA-West_Point/Part_Collection)

- [Safety](https://2017.igem.org/Team%3AUSMA-West_Point/Safety)
- Human Practices
- [Integrated and Gold](https://2017.igem.org/Team%3AUSMA-West_Point/HP/Gold_Integrated)
- [Public Engagement](https://2017.igem.org/Team%3AUSMA-West_Point/Engagement)

- Awards
- [Entreneurship](https://2017.igem.org/Team%3AUSMA-West_Point/Entrepreneurship)
- [Applied design](https://2017.igem.org/Team%3AUSMA-West_Point/Applied_Design)
- [Hardware](https://2017.igem.org/Team%3AUSMA-West_Point/Hardware)

# Results

## Plasmid Design and Development

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.

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 (Below): 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).

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.

## Animal Cell Nucleofection

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.

## Western Blot

## Immunohistochemistry

## Physiological Response

## Handheld Photosensor Device

## Microelectrode Array Bioreactor

## DEPARTMENT OF CHEMISTRY AND LIFE SCIENCE

![](https://lh3.googleusercontent.com/EWqW7DEI4kOTRMLjK2-ObFHp-EYBt5apFYZ1LVFAhLtTLjigCRfx5hCCTKbIjIm68VQ00p9twloHJ9w8=s50)
![](https://lh3.googleusercontent.com/TgRyMQvJ3_h9RmOnu7AlhIE7NLOOBsRoBounARrs8fQv8HCRPaFtpBneSqJOSZpI6l7He_bAZKN179JBig=s50)
![](https://lh3.googleusercontent.com/43-pXHjwrpmVO8Oean-6BD0uzARvcqUQrpdi7Yw2bxaXwEoP21UdN5kW6Ks9pdOxf7ropMUrh0djgYPwYPU=s50)
![](https://lh3.googleusercontent.com/9rwgVnDglPdPFugSu98fhDmxzjXC9KovZ_7BuHkXPIv6jvg9S96flGnhL_e4y8mIpPpZQstfqEV-WitY=s50)

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