iGEM 2018 — Team USMA-West_Point

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# Team:USMA-West Point/Notebook

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TEAM

[Team Members](https://2018.igem.org/Team%3AUSMA-West_Point/Team)
[Collaborations](https://2018.igem.org/Team%3AUSMA-West_Point/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AUSMA-West_Point/Description)
[Design](https://2018.igem.org/Team%3AUSMA-West_Point/Design)
[Experiments](https://2018.igem.org/Team%3AUSMA-West_Point/Experiments)
[Notebook](https://2018.igem.org/Team%3AUSMA-West_Point/Notebook)
[InterLab](https://2018.igem.org/Team%3AUSMA-West_Point/InterLab)
[Model](https://2018.igem.org/Team%3AUSMA-West_Point/Model)
[Results](https://2018.igem.org/Team%3AUSMA-West_Point/Results)
[Demonstrate](https://2018.igem.org/Team%3AUSMA-West_Point/Demonstrate)
[Improve](https://2018.igem.org/Team%3AUSMA-West_Point/Improve)
[Attributions](https://2018.igem.org/Team%3AUSMA-West_Point/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AUSMA-West_Point/Parts)
[Basic Parts](https://2018.igem.org/Team%3AUSMA-West_Point/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AUSMA-West_Point/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AUSMA-West_Point/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AUSMA-West_Point/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AUSMA-West_Point/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AUSMA-West_Point/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AUSMA-West_Point/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AUSMA-West_Point/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AUSMA-West_Point/Hardware)
[Measurement](https://2018.igem.org/Team%3AUSMA-West_Point/Measurement)
[Model](https://2018.igem.org/Team%3AUSMA-West_Point/Model)
[Plant](https://2018.igem.org/Team%3AUSMA-West_Point/Plant)
[Software](https://2018.igem.org/Team%3AUSMA-West_Point/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=USMA-West_Point)

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# **Notebook**

**May 2018**

* Presentation of overall project to develop cell-based artificial chemosensory detectors at MIT Solider Design Competition

**June 2018**

* Started design for synthetic operon plasmid

**July 2018**

* Submitted design for synthetic operon plasmid to GeneWiz for synthesis
* Interlab studies completed

**August 2018**

* Designed primers for hOR1D2 expression plasmid
* Amplified hOR1D2 open reading frame by PCR

**September 2018**

* Established collaboration with USAFA iGEM team
* Started analysis of USAFA iGEM plasmid intended for DNA part submission
* Digested hOR1D2 PCR product and pET21a expression vector
* Ligation reactions for inserting hOR1D2 into pET21a vector, and transformations of DH5alpha cells with ligation reactions

**October 2018**

* Completed analysis of USAFA iGEM plasmid intended for DNA part submission
* Cadet McDonough discussed human practices considerations with Col. Barnes, USMA Dept. of English and Philosophy
* Took delivery of synthetic operon plasmid from GeneWiz
* Analysis of pET21A plasmid containing hOR1D2 insert
* Transform BL21(DE3) cells with hOR1D2/pET21a and synthetic operon plasmids
* Transform BL21(DE3) cells with synthetic operon plasmid
* Attend Jamboree

---

### USMA-West Point

Team USMA-West Point is the United States Military Academy's iGEM team, representing the Department of Chemistry and Life Science.

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### USMA's Department of Chemistry and Life Science

* ![](/wiki/images/a/a5/T--USMA-West_Point--logo.jpeg)
  [USMA Home Page](https://www.usma.edu/SitePages/Home.aspx)

- ![](/wiki/images/d/dc/T--USMA-West_Point--CLS.jpeg)
  [USMA's Department of Chemistry and Life Science Facebook](https://www.facebook.com/West-Point-Department-of-Chemistry-and-Life-Science-385389195808/)


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# Team:USMA-West Point/Experiments

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TEAM

[Team Members](https://2018.igem.org/Team%3AUSMA-West_Point/Team)
[Collaborations](https://2018.igem.org/Team%3AUSMA-West_Point/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AUSMA-West_Point/Description)
[Design](https://2018.igem.org/Team%3AUSMA-West_Point/Design)
[Experiments](https://2018.igem.org/Team%3AUSMA-West_Point/Experiments)
[Notebook](https://2018.igem.org/Team%3AUSMA-West_Point/Notebook)
[InterLab](https://2018.igem.org/Team%3AUSMA-West_Point/InterLab)
[Model](https://2018.igem.org/Team%3AUSMA-West_Point/Model)
[Results](https://2018.igem.org/Team%3AUSMA-West_Point/Results)
[Demonstrate](https://2018.igem.org/Team%3AUSMA-West_Point/Demonstrate)
[Improve](https://2018.igem.org/Team%3AUSMA-West_Point/Improve)
[Attributions](https://2018.igem.org/Team%3AUSMA-West_Point/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AUSMA-West_Point/Parts)
[Basic Parts](https://2018.igem.org/Team%3AUSMA-West_Point/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AUSMA-West_Point/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AUSMA-West_Point/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AUSMA-West_Point/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AUSMA-West_Point/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AUSMA-West_Point/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AUSMA-West_Point/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AUSMA-West_Point/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AUSMA-West_Point/Hardware)
[Measurement](https://2018.igem.org/Team%3AUSMA-West_Point/Measurement)
[Model](https://2018.igem.org/Team%3AUSMA-West_Point/Model)
[Plant](https://2018.igem.org/Team%3AUSMA-West_Point/Plant)
[Software](https://2018.igem.org/Team%3AUSMA-West_Point/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=USMA-West_Point)

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# **Project**

## **Experiments**

**Plasmid Purification**

5mL LB cultures were inoculated and grown overnight at 37C and shaking at 220rpm

Cultures were spun at 3700rcf for 5min, and supernatant was discarded

Plasmids were isolated using the DNA Plasmid Miniprep Kit (Qiagen)

**PCR**

25uL 2X PCR Taq Plus Mastermix (ABM Bioscience)

1uL template plasmid

1uL forward primer (final concentration 0.5uM)

1uL reverse primer (final concentration 0.5uM)

22uL water

Reactions were run on BioRad MyCycler thermocycler with the following protocol:

Step 1: 95C for 1min

Step 2: 95C for 30sec

Step 3: 52C for 30sec

Step 4: 71C for 30sec

Repeat steps 2 through 4 for 40 cycles

Step 5: 71C for 2min

Step 6: hold at 4C

The generation of PCR products was confirmed using agarose gel electrophoresis

**PCR reaction clean up**

PCR reactions were purified using the PCR Clean-up Kit (Qiagen) following the manufacturer’s instructions

**Restriction Digest Reactions**

2uL 10X reaction digest buffer (New England BioLabs)

1uL enzyme 1 (New England BioLabs)

1uL enzyme 2 (New England BioLabs)

1-10uL Plasmid or PCR insert

Adjust with water so that final volume is 20uL

All reactions were run overnight at 37C in a BioRad MyCycler thermocycler

Prior to reaction clean up, plasmids were treated with 1uL Antarctic Alkaline Phosphatase (New England BioLabs) for 1hr at 37C

All reactions were cleaned up with PCR Clean-up Kit (Qiagen) following the manufacturer’s instructions

**Ligation Reactions**

2uL 10X T4 Ligase Buffer (New England BioLabs)

0.5uL T4 DNA Ligase (New England BioLabs)

1-5uL of digested target plasmid (200ng of plasmid)

5-12.5uL of digested insert (600ng of insert)

Adjust volume with water to 20uL

Ligation reactions were incubated overnight at 16C a BioRad MyCycler thermocycler

**Bacterial Transformations**

Competent DH5alpha cells or BL21(DE3) cells (New England BioLabs) were thawed on ice

5uL of ligation reaction was added to cells and stirred

Cells/ligation solution was incubated on ice for 20min

Cells/ligation solution was placed in 37C heat block for 30sec

Cells/ligation solution were placed back on ice for an additional 2min

Cells/ligation solution were transferred to 100uL of pre-warmed SOC media

Cells in SOC media were incubated at 37C with shaking at 220rpm for 1hr

Cells were then plated on LB/Agar plates containing the appropriate antibiotic

Plates were placed in a 37C bacterial incubator at 37C overnight

Plates were removed in the morning

**Colony screening**

Individual colonies were used to inoculate 5mL LB cultures

Cultures were grown overnight

Plasmids were isolated using the DNA Plasmid Miniprep Kit (Qiagen) following the manufacturer’s protocol

Plasmids were then digested with restriction enzyme reactions at 37C overnight in 16C a BioRad MyCycler thermocycler

Reaction products were analyzed by gel electrophoresis

**Agarose Gel Electrophoresis**

0.8g of Agarose (Sigma) was dissolved in 100mL of 1X TAE (Sigma)

Agarose solution was microwaved until boiling

Molten agarose solution was used to cast gel

0.5uL of ethidium bromide stock solution was added to the molten agarose and stirred thoroughly

Gel comb was placed in mold

Gel was allowed to solidify at room temperature before being transferred to gel running box

1X TAE running buffer was added until gel was submerged

*Sample prep*

*Samples:*

5uL of PCR reactions were loaded straight into gels (PCR buffer is designed for straight-to-gel analysis)

For all other samples, 4uL of 6X loading buffer (New England BioLabs) was added per 20uL volume

*Ladders:*

1uL 100bp or 1kb ladder (New England BioLabs)

2uL 6X loading buffer (New England BioLabs)

9uL water

Samples were loaded into respective wells

Gel was run at ~100V for ~40min

Bands on gel were visualized using a transilluminator (UVP)

**Expression of OR1D2 and Synthetic Operon Proteins**

Overnight cultures in minimal media using glycerol as the carbon source were inoculated with BL21(DE3) bacteria co-transformed with hOR1D2 and synthetic operon expression plasmids. Overnight cultures were incubated at 37C with shaking at 220rpm. The following morning, overnight culture was used to inoculate 4 fresh cultures in 20mL minimal media to an OD600 of 0.2. Cultures were grown incubated at 37C with shaking at 220rpm until the OD600 reached 0.6, when cultures were pelleted by centrifugation (3500xg for 5min), resuspended in PBS and then pelleted again by centrifugation (3500xg for 5min), before being resuspended with minimal containing either arabinose with IPTG (media had no glycerol), arabinose only (media had no glycerol), IPTG (media contained glycerol) only, or water (media contained glycerol). Arabinose had a final concentration of 0.1% and IPTG had a final concentration of 0.5mM. After the media change and addition of either arabinose and/or IPTG, cultures were incubated at 37C with shaking at 220rpm for an additional 3 hours, after which cells were pelleted by centrifugation (3500xg for 5min) for RNA harvest and qPCR analysis.

**Collection of RNA**

Cells were pelleted by centrifuging cultures at 3500xg for 5min, and supernatant discarded

RNA was isolated using RNA Miniprep Isolated Kit (Qiagen) following manufacturer’s instructions

**cDNA First Strand Synthesis**

1-8uL of RNA (3ug RNA)

1uL Random hexamer primers (ThermoFisher)

1uL 10mM dNTPs (ThermoFisher)

Volume adjusted to 10uL with DEPC-treated water

RNA/NTP solution was incubated at 65C for 5 in a BioRad MyCycler thermocycler, then the following were added:

2uL 10XRT buffer (ThermoFisher)

4uL 25mM MgCl2 (ThermoFisher)

2uL 0.1M DTT (ThemroFisher)

1uL RNaseOUT (ThermoFisher)

1uL SuperScript (ThermoFisher)

Reactions (20uL total volume) were run in BioRad MyCycler thermocycler at:

25C for 10min

50C for 50min

85C for 5min

**qPCR**

All samples were run in triplicate

Individual samples were prepared as follows:

0.3uL cDNA

0.5uL forward primer

0.5uL reverse primer

10uL 2XSYBR Green Dye

8.7uL Water

Reactions were placed in a 96well qPCR plate (Applied Biosystems) and sealed with microplate adhesive film (Applied Biosystems)

Reactions were run on an Applied Biosystem 7500 Real Time PCR system

Data was analyzed using Excel (Microsoft)

---

### USMA-West Point

Team USMA-West Point is the United States Military Academy's iGEM team, representing the Department of Chemistry and Life Science.

Powered by [w3.css](https://www.w3schools.com/w3css/default.asp)

### USMA's Department of Chemistry and Life Science

* ![](/wiki/images/a/a5/T--USMA-West_Point--logo.jpeg)
  [USMA Home Page](https://www.usma.edu/SitePages/Home.aspx)

- ![](/wiki/images/d/dc/T--USMA-West_Point--CLS.jpeg)
  [USMA's Department of Chemistry and Life Science Facebook](https://www.facebook.com/West-Point-Department-of-Chemistry-and-Life-Science-385389195808/)

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# Team:USMA-West Point/Results

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[HOME](https://2018.igem.org/Team%3AUSMA-West_Point)

TEAM

[Team Members](https://2018.igem.org/Team%3AUSMA-West_Point/Team)
[Collaborations](https://2018.igem.org/Team%3AUSMA-West_Point/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AUSMA-West_Point/Description)
[Design](https://2018.igem.org/Team%3AUSMA-West_Point/Design)
[Experiments](https://2018.igem.org/Team%3AUSMA-West_Point/Experiments)
[Notebook](https://2018.igem.org/Team%3AUSMA-West_Point/Notebook)
[InterLab](https://2018.igem.org/Team%3AUSMA-West_Point/InterLab)
[Model](https://2018.igem.org/Team%3AUSMA-West_Point/Model)
[Results](https://2018.igem.org/Team%3AUSMA-West_Point/Results)
[Demonstrate](https://2018.igem.org/Team%3AUSMA-West_Point/Demonstrate)
[Improve](https://2018.igem.org/Team%3AUSMA-West_Point/Improve)
[Attributions](https://2018.igem.org/Team%3AUSMA-West_Point/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AUSMA-West_Point/Parts)
[Basic Parts](https://2018.igem.org/Team%3AUSMA-West_Point/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AUSMA-West_Point/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AUSMA-West_Point/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AUSMA-West_Point/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AUSMA-West_Point/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AUSMA-West_Point/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AUSMA-West_Point/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AUSMA-West_Point/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AUSMA-West_Point/Hardware)
[Measurement](https://2018.igem.org/Team%3AUSMA-West_Point/Measurement)
[Model](https://2018.igem.org/Team%3AUSMA-West_Point/Model)
[Plant](https://2018.igem.org/Team%3AUSMA-West_Point/Plant)
[Software](https://2018.igem.org/Team%3AUSMA-West_Point/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=USMA-West_Point)

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# **Project**

## Results

*Construction of hOR1D2 expression plasmid*

A mammalian expression plasmid containing hOR1D2 was previously obtained by the laboratory for other eNOSE-related projects. Since this project requires hOR1D2 expression in bacteria, however, the hOR1D2 coding sequence needed to be shuttled to a bacterial expression plasmid, the pET-21A expression vector. To amplify the hOR1D2 coding with restriction enzyme sites compatible for the pET-21A vector, the following oligonucleotides were ordered (from ThermoFisher):

Forward primer: 5’-ATCGATCG GAATTC **ATG**GATGGAGGCAACCAGAGT-3’

Reverse primer: 5’- ATCGATCG AAGCTT **TCA**TGTCAGCCTCTTAAAGTGTTTATCT-3’

The primers were designed to place an EcoRI restriction enzyme site on the 5’ end of the coding sequence (EcoRI site is underlined in the forward primer sequence; ATG translation initiation codon is in bold). The primers were also designed to introduce a HindIII restriction enzyme site on the 3’ end of the coding sequence (HindIII site is underlined in the reverse primer sequence;  translation Stop codon is in bold).

Together, these primers were designed to directionally orient the hOR1D2 coding sequence in frame with the open reading frame established in the pET-21A plasmid (see Figure 1).

![Steak](/wiki/images/c/cf/T--USMA-West_Point--pet.jpg)

**Figure 1**: Multiple cloning site and open reading frame for pET-21A plasmid (figure source Nova Lifetech Inc).

Following the PCR reaction to amplify hOR1D2 coding sequence with the primers shown above, an agarose gel was run to confirm amplification of the coding sequence (Figure 2). This gel shows a single band at just under 1kb, which is consistent with the predicted 967bp length based on sequence information.

![Steak](/wiki/images/c/c5/T--USMA-West_Point--hor.jpg)

**Figure 2**: Amplification of the hOR1D2 coding sequence by PCR

This PCR product was purified, and then digested with EcoRI and HindIII overnight. The following morning, the plasmid was purified from digestion reaction, and eluted in a 30uL volume of water. The NanoDrop measured that the purified, digested hOR1D2 insert was at 55ng/uL.

A 1uL aliquot of 250ng/uL stock of pET-21A was used to transform DH5alpha E.coli. A single colony from this transformation was used to start an overnight 5mL culture. A half milliliter of this overnight culture was used to make glycerol stock that was stored in the -80C freezer, and the remaining portion of the culture was used to produce a 50uL stock of pET-21A in water at 400ng/uL.

To prepare the pET-21A, 5ug was digested with EcoRI and HindIII overnight. The following morning, 1uL of Antarctic Alkaline Phosphatase was added to the digestion reaction (to dephosphorylate free 5’ phosphate groups on the digested plasmid and reduce background colonies), and incubated for an additional hour, after which the reaction was purified into 30uL of water. The NanoDrop measured that the purified, digested plasmid was at 145ng/uL.

The digested hOR1D2 insert and pET-21A plasmid were ligated using 450ng of insert and 145ng of plasmid (aiming for the recommended 3:1 ratio of insert:plasmid for ligations). Ligation reactions were run overnight at 16C and then used to transform DH5alpha E.coli that were plated on LB/Agar plates containing 100ug/mL ampicillin.

The transformation unexpectedly produced a single colony. After some discussion, the competent DH5alpha cells used for this transformation were identified as cells from a batch that may have been inadvertently thawed over the summer when the -80C freezer it was originally stored in was emptied for maintenance and cleaning. This thawing would have certainly compromised the competence of the cells. This single colony was used to inoculate an overnight culture and the plasmid was isolated by miniprep (50uL of 860ng/uL).

To verify ligation of the hOR1D2 insert into pET-21A, the isolated plasmid was digested by EcoRI and HindIII for 3 hours before being analyzed on an agarose gel. If the plasmid is correctly constructed, this digest will generate a fragment that are ~950bp and ~5.4kb (Figure 3A). The gel confirmed production of a ~950bp fragment, but the second fragment was at ~3.5kb instead of the anticipated ~5.4kb (Figure 3C). A second digest with NdeI was expected to generate fragment of ~400bp and ~6kb (Figure 3B). This NdeI digest, however, produced bands at ~700bp and ~4kb. At the time of the iGEM Wiki freeze, the discrepancies for these differences in the expected molecular weights of the digests was still being discussed and examined. Since the PCR reaction amplifying the hOR1D2 coding sequence worked as anticipated, we concentrate on the whether there is an issue with the plasmid (e.g.: perhaps a mislabeled stock was used?) and it is expected that the ligation reactions will be redone.

![Steak](/wiki/images/3/31/T--USMA-West_Point--analysis.jpg)

**Figure 3**: Analysis of hOR1D2 in pET-21A clone.

*Construction of synthetic operon plasmid*

An oligonucleotide was designed for generating the synthetic operon. This operon was designed to use the AraC-pBad promoter sequence that is identical to the AracC-Pbad promoter that is available in the iGEM parts database (part BBa\_K808000). The coding regions for the His-tagged human Tau and Strep-tagged icaB genes were placed downstream of this promoter. Both genes were designed for E.coli codon use bias.

The oligonucleotide sequence for this synthetic operon was initially sent to IDT for its construction. The initial estimate of 4-6 weeks for its delivery, but after several months it was determined that the oligonucleotide could not be constructed as designed. An alternative vendor, GeneWiz, was contacted and offered to complete the construction and incorporate the operon into a pUC57 plasmid backbone. This construction was also reported several challenges that resulted in the multiple delays in the projected completion date. By the end of September, however, GeneWiz reported successful construction of a sequence-verified vector containing our synthetic operon.

Upon taking delivery of the synthetic operon plasmid, the plasmid was used to transform DH5alpha cells on LB/agar plates with 50ug/mL kanamycin. These colonies were used inoculate overnight growth generate a glycerol stock and plasmid stock (50uL at 900ug/uL). The plasmid has also been used to transform BL21(DE3) cells also on LB/agar plates with 50ug/mL kanamycin.

*Future Directions*

The BL21(DE3) cells transformed with the synthetic operon will be used to establish that the His-tagged human Tau and Strep-tagged icaB genes can be induced with arabinose. These experiments will grow cells on minimal media with glycerol as a carbon source, which should suppress expression of the operon genes. When the carbon source is switched to arabinose, however, the expression of the operon genes should be induced. This expression will be verified by qPCR and western blotting.

Once the construction issues for hOR1D2 in pET-21A have been resolved, this plasmid will be used with the synthetic operon plasmid to co-transform BL21(DE3) cells. These co-transformed cells will be used to test whether both the operon and hOR1D2 genes can be simultaneous co-expressed. This experiment be performed in the same way as the operon-only studies (see above), but in addition to arabinose, IPTG will also be added since this will be required to drive expression from the pET-21A plasmid. The co-expression of the operon and OR1D2 genes will also be verified by qPCR and western blotting.

---

### USMA-West Point

Team USMA-West Point is the United States Military Academy's iGEM team, representing the Department of Chemistry and Life Science.

Powered by [w3.css](https://www.w3schools.com/w3css/default.asp)

### USMA's Department of Chemistry and Life Science

* ![](/wiki/images/a/a5/T--USMA-West_Point--logo.jpeg)
  [USMA Home Page](https://www.usma.edu/SitePages/Home.aspx)
* ![](/wiki/images/d/dc/T--USMA-West_Point--CLS.jpeg)
  [USMA's Department of Chemistry and Life Science Facebook](https://www.facebook.com/West-Point-Department-of-Chemistry-and-Life-Science-385389195808/)