iGEM 2019 — Team Queens_Canada

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Queens Canada/Notebook

[QGEM](http://queensigem.ca/)

* [Home (current)](https://2019.igem.org/Team%3AQueens_Canada)
* Project

  [Description](https://2019.igem.org/Team%3AQueens_Canada/Description)
  [Background](https://2019.igem.org/Team%3AQueens_Canada/Background)
  [Demonstrate](https://2019.igem.org/Team%3AQueens_Canada/Demonstrate)
* Team

  [Meet the Team](https://2019.igem.org/Team%3AQueens_Canada/Team)
  [Attributions](https://2019.igem.org/Team%3AQueens_Canada/Attributions)
  [Sponsors](https://2019.igem.org/Team%3AQueens_Canada/Sponsors)
* Laboratory

  [Overview](https://2019.igem.org/Team%3AQueens_Canada/Lab_Overview)
  [Design](https://2019.igem.org/Team%3AQueens_Canada/Design)
  [Model](https://2019.igem.org/Team%3AQueens_Canada/Model)
  [Experiments](https://2019.igem.org/Team%3AQueens_Canada/Experiments)
  [Results](https://2019.igem.org/Team%3AQueens_Canada/Results)

  [Safety](https://2019.igem.org/Team%3AQueens_Canada/Safety)
  [Notebook](https://2019.igem.org/Team%3AQueens_Canada/Notebook)
* Parts

  [Overview](https://2019.igem.org/Team%3AQueens_Canada/Parts)
  [Basic Parts](https://2019.igem.org/Team%3AQueens_Canada/Basic_Part)
  [Contribution](https://2019.igem.org/Team%3AQueens_Canada/Contribution)
* Prototype

  [Overview](https://2019.igem.org/Team%3AQueens_Canada/Prototype_Overview)
  [Fluorometer](https://2019.igem.org/Team%3AQueens_Canada/Fluorometer)
  [Software](https://2019.igem.org/Team%3AQueens_Canada/Software)
  [Hardware](https://2019.igem.org/Team%3AQueens_Canada/Hardware)
* Human Practices

  [Integrated HP](https://2019.igem.org/Team%3AQueens_Canada/Human_Practices)
  [Public Engagement](https://2019.igem.org/Team%3AQueens_Canada/Public_Engagement)
  [Entrepreneurship](https://2019.igem.org/Team%3AQueens_Canada/Entrepreneurship)
* [Collaborations](https://2019.igem.org/Team%3AQueens_Canada/Collaborations)

* Search

![](https://static.igem.org/mediawiki/2019/9/93/T--Queens_Canada--Laboratory_Notebook.png)

Week 1

Finalized project design and approach

Week 2

2 Biobrick designs finished.

Week 3

Drylab started basic prototype design -- arduino board was linked to an LED circuit. The Outreach team started Synthetic biology information sessions about synthetic biology, for students at Queen's University.

Week 4

BioBricks arrived from IDT and were cloned into a pETDuet-1 vector.
Dry lab set up a circuit with a photoresistor that links to the LED. Additionally, the team attending training sessions on 3-D printing at SparQ Studios.

Week 5

Top10 cells transformed with the BioBricks were Mini-prepped and checked with a test-Digest.The transformations were successful. Dry-lab started testing the arduino light sensor sensitivity using a GFP stock. Additionally, the Dry-lab team made a CAD model and valve system for the device. Outreach held another synthetic biology information session and started team logo design.

Week 6

Purified plasmids containing the BioBricks were sequenced and then transformed into BL21 cells, for expression. Dry-lab made the first 3D model of the prototype and collected more data on the light sensor sensitivity. Outreach set up meetings with the Kingston Police. Additionally, the first logo idea was proposed and Wiki design started.

Week 7

Carried out a small scale purification on the BL21 cells expressing the BioBricks. However, purification was unsuccessful. Dry-lab finished the first custom PCB design for the prototype and improved on the fluid distribution system. Outreach designed the second iteration of the team logo and organized a lesson plan for attending Science Quest. Science QUest is an educational summer camp at Queen's University.

Week 8

Designed and tested a protocol for determining affinity of antibodies to lipophilic antigens. Protocol was used to determine antibody affinity for THC. The wet-lab also re-expressed the biobricks in BL21; however, no fluorescence was detected, indicating failed expression. Dry lab edited circuit to include portable power source and improved the CAD model.

Week 9

Concern about current vector was found and started amplifying BioBricks out of pETDuet-1, in order to transform them into pET24d and pET16b. Ordered re-designed biobricks with varying fluorescent proteins. Dry-lab started building a java application for the prototype. Our team met with a Drug Recognition expert from the Kingston Police to provide advice on product design.

Week 10

One of the BioBricks, a ScFv ligated to mNG was ligated into a pET24d vector. Product was transformed into Top10 cells. Dry-lab designed a bluetooth module for the prototype, based on the feedback given by the Kingston Police. Outreach team taught a lesson on synthetic biology during Science Quest, at Queen's University.

Week 11

Transformed the previous ScFv-mNG biobrick into BL21 for expression. A test expression did not show fluorescence under a confocal microscope. Dry-lab finalized the custom PCB and placed an order. Outreach set up a lesson plan with Steamworks (Kingston, ON), to teach the community about synthetic biology.

Week 12

Transformed an Fab biobrick into BL21 for expression. Dry-lab designed and printed parts for the tubing inside the prototype. Outreach has connected with the Canadian Research Initiative of Substance Abuse (CRISM), to gain information on Cannabis usage and legislation.

Week 13

Performed a small scale expression for BioBricks. Redesigned the PCB and CAD model, to better fit device components. Wiki tabs were designed.

Week 14

Performed a small scale purification of the antibodies produced by the BioBricks. Dry-lab improved bluetooth communication with devices. Outreach started a video series on tackling the most asked questions in synthetic biology.

Week 15

Performed a small scale purification of the antibodies produced by the BioBricks. Dry-lab improved bluetooth communication with devices. Outreach started a video series on tackling the most asked questions in synthetic biology.

Week 16

Used purified proteins on a THC membrane detection assay and discovered that the ScFv-mNG bound THC and gave off a fluorescent signal! Dry-lab made a new CAD design to better fit components.

Week 17

Performed a large scale purification to obtain more protein for the membrane assays.

Week 18

Membrane assay were repeated with to determine detection limit of THC.

Week 19

Repeated membrane assays and tried to adapt detection protocol for usage in 96 well plates. 96 well plates were not suitable for THC detection, as THC did not bind the wells.

Week 20

Optimized time-scale of membrane assay and managed to reduce protocol to 30 minutes.

![](https://static.igem.org/mediawiki/2019/f/f7/T--Queens_Canada--Home_footer.png)

[![](https://static.igem.org/mediawiki/2019/b/b6/T--Queens_Canada--QGEM_name.png)](http://queensigem.ca/)

Queen's Genetically Engineered Machine Team

Contact us at: qgem@engsoc.queensu.ca

Follow us on:

[![](https://static.igem.org/mediawiki/2019/9/98/T--Queens_Canada--Facebook_logo.png)](https://www.facebook.com/iGEMQueens)
[![](https://static.igem.org/mediawiki/2019/e/e7/T--Queens_Canada--Instagram_logo.png)](https://www.instagram.com/queens_igem/)

[![](https://static.igem.org/mediawiki/2019/9/98/T--Queens_Canada--Twitter_logo.png)](https://twitter.com/igemqueens)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Queens Canada/Experiments

[QGEM](http://queensigem.ca/)

* [Home (current)](https://2019.igem.org/Team%3AQueens_Canada)
* Project

  [Description](https://2019.igem.org/Team%3AQueens_Canada/Description)
  [Background](https://2019.igem.org/Team%3AQueens_Canada/Background)
  [Demonstrate](https://2019.igem.org/Team%3AQueens_Canada/Demonstrate)
* Team

  [Meet the Team](https://2019.igem.org/Team%3AQueens_Canada/Team)
  [Attributions](https://2019.igem.org/Team%3AQueens_Canada/Attributions)
  [Sponsors](https://2019.igem.org/Team%3AQueens_Canada/Sponsors)
* Laboratory

  [Overview](https://2019.igem.org/Team%3AQueens_Canada/Lab_Overview)
  [Design](https://2019.igem.org/Team%3AQueens_Canada/Design)
  [Model](https://2019.igem.org/Team%3AQueens_Canada/Model)
  [Experiments](https://2019.igem.org/Team%3AQueens_Canada/Experiments)
  [Results](https://2019.igem.org/Team%3AQueens_Canada/Results)

  [Safety](https://2019.igem.org/Team%3AQueens_Canada/Safety)
  [Notebook](https://2019.igem.org/Team%3AQueens_Canada/Notebook)
* Parts

  [Overview](https://2019.igem.org/Team%3AQueens_Canada/Parts)
  [Basic Parts](https://2019.igem.org/Team%3AQueens_Canada/Basic_Part)
  [Contribution](https://2019.igem.org/Team%3AQueens_Canada/Contribution)
* Prototype

  [Overview](https://2019.igem.org/Team%3AQueens_Canada/Prototype_Overview)
  [Fluorometer](https://2019.igem.org/Team%3AQueens_Canada/Fluorometer)
  [Software](https://2019.igem.org/Team%3AQueens_Canada/Software)
  [Hardware](https://2019.igem.org/Team%3AQueens_Canada/Hardware)
* Human Practices

  [Integrated HP](https://2019.igem.org/Team%3AQueens_Canada/Human_Practices)
  [Public Engagement](https://2019.igem.org/Team%3AQueens_Canada/Public_Engagement)
  [Entrepreneurship](https://2019.igem.org/Team%3AQueens_Canada/Entrepreneurship)
* [Collaborations](https://2019.igem.org/Team%3AQueens_Canada/Collaborations)

* Search

![](https://static.igem.org/mediawiki/2019/0/04/T--Queens_Canada--Laboratory_Experiments.png)

### Overview

Laboratory experiments were conducted in tandem with the hardware and software design. The progression can be summed up as follows:
**1)** Designing a one-step membrane assay for THC detection, using lipophilic membranes and a commercial anti-THC antibody
**2)** Modeling of single chain variable fragments (ScFv) and antigen binding fragments (Fab), to link them to fluorescent proteins
**3)** Using the models to design a library of ScFv and Fab, linked to fluorescent proteins
**4)** Cloning into pET vectors for high protein expression
**5)** Testing proteins for THC binding ability on novel one-step membrane assay
**6)** Testing antibody binding limit using one-step membrane assay

### Links to Protocols

[Novel Immunoassay for lipophilic antigen detection](https://www.protocols.io/view/novel-immuno-assay-for-detection-of-lipophilic-sub-8bghsjw?full_view_mode)
[Diagnostic Restriction Digest](https://2018.igem.org/Team%3AQueens_Canada/Diagnostic-Restriction-Digest)
[gBlock Gibson Assembly](https://2018.igem.org/Team%3AQueens_Canada/gibson)
[Glycerol Stocks](https://2018.igem.org/Team%3AQueens_Canada/gly)
[His-Tag Nickel Chromatography](https://2018.igem.org/Team%3AQueens_Canada/nick)
[LB Broth and Agar Plates](https://2018.igem.org/Team%3AQueens_Canada/agar)
[Liquid Culture](https://2018.igem.org/Team%3AQueens_Canada/liquid)
[Lysis by Sonication](https://2018.igem.org/Team%3AQueens_Canada/lysis)
[MiniPrep](https://2018.igem.org/Team%3AQueens_Canada/mini)
[Resuspension of gBlocks Gene Fragment](https://2018.igem.org/Team%3AQueens_Canada/gblocks)
[Streaking Plates](https://2018.igem.org/Team%3AQueens_Canada/streak)

![](https://static.igem.org/mediawiki/2019/f/f7/T--Queens_Canada--Home_footer.png)

[![](https://static.igem.org/mediawiki/2019/b/b6/T--Queens_Canada--QGEM_name.png)](http://queensigem.ca/)

Queen's Genetically Engineered Machine Team

Contact us at: qgem@engsoc.queensu.ca

Follow us on:

[![](https://static.igem.org/mediawiki/2019/9/98/T--Queens_Canada--Facebook_logo.png)](https://www.facebook.com/iGEMQueens)
[![](https://static.igem.org/mediawiki/2019/e/e7/T--Queens_Canada--Instagram_logo.png)](https://www.instagram.com/queens_igem/)

[![](https://static.igem.org/mediawiki/2019/9/98/T--Queens_Canada--Twitter_logo.png)](https://twitter.com/igemqueens)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Queens Canada/Results

[QGEM](http://queensigem.ca/)

* [Home (current)](https://2019.igem.org/Team%3AQueens_Canada)
* Project

  [Description](https://2019.igem.org/Team%3AQueens_Canada/Description)
  [Background](https://2019.igem.org/Team%3AQueens_Canada/Background)
  [Demonstrate](https://2019.igem.org/Team%3AQueens_Canada/Demonstrate)
* Team

  [Meet the Team](https://2019.igem.org/Team%3AQueens_Canada/Team)
  [Attributions](https://2019.igem.org/Team%3AQueens_Canada/Attributions)
  [Sponsors](https://2019.igem.org/Team%3AQueens_Canada/Sponsors)
* Laboratory

  [Overview](https://2019.igem.org/Team%3AQueens_Canada/Lab_Overview)
  [Design](https://2019.igem.org/Team%3AQueens_Canada/Design)
  [Model](https://2019.igem.org/Team%3AQueens_Canada/Model)
  [Experiments](https://2019.igem.org/Team%3AQueens_Canada/Experiments)
  [Results](https://2019.igem.org/Team%3AQueens_Canada/Results)

  [Safety](https://2019.igem.org/Team%3AQueens_Canada/Safety)
  [Notebook](https://2019.igem.org/Team%3AQueens_Canada/Notebook)
* Parts

  [Overview](https://2019.igem.org/Team%3AQueens_Canada/Parts)
  [Basic Parts](https://2019.igem.org/Team%3AQueens_Canada/Basic_Part)
  [Contribution](https://2019.igem.org/Team%3AQueens_Canada/Contribution)
* Prototype

  [Overview](https://2019.igem.org/Team%3AQueens_Canada/Prototype_Overview)
  [Fluorometer](https://2019.igem.org/Team%3AQueens_Canada/Fluorometer)
  [Software](https://2019.igem.org/Team%3AQueens_Canada/Software)
  [Hardware](https://2019.igem.org/Team%3AQueens_Canada/Hardware)
* Human Practices

  [Integrated HP](https://2019.igem.org/Team%3AQueens_Canada/Human_Practices)
  [Public Engagement](https://2019.igem.org/Team%3AQueens_Canada/Public_Engagement)
  [Entrepreneurship](https://2019.igem.org/Team%3AQueens_Canada/Entrepreneurship)
* [Collaborations](https://2019.igem.org/Team%3AQueens_Canada/Collaborations)

* Search

![](https://static.igem.org/mediawiki/2019/1/10/T--Queens_Canada--Lab_Results.png)

### Cloning

The biobrick was ordered from IDT, digested, and ligated into pET24d (Fig. 1). pET24d was chosen as a vector due to the availability of the T7 promoter.

![](https://static.igem.org/mediawiki/parts/e/ed/T--Queens_Canada--ScFv-mNG_Digest.jpg)

Figure 1. Test digest of ScFv-mNG integration into the pET24d vector. The band above 1.5 kb indicates successful integration of the 1536 bp biobrick.

### Expression

The biobrick was expressed in E. coli Rosetta gami2, as this cell line contains an oxidizing cytoplasmic environment ideal for the disulfide bond formations within the antibody (Fig. 2). Following a nickle affinity column purification, the ScFv was observed at 56 kDa. Note that another biobrick (BBa\_K3056001) was designed, with EGFP as the fluorescent protein, rather than mNG, to compare fluorescent intensities.

![](https://static.igem.org/mediawiki/parts/5/5f/T--Queens_Canada--ScFv_expression.jpg)

Figure 2. Nickel column purification of ScFv-mNG and ScFv-EGFP. Both proteins were successfully purified from BL21 cells, as indicated by the major band at 56 kDa.

### THC Binding

The purified EGFP and mNG linked ScFv were spot tested on a membrane assay, where only the ScFv was able to bind the THC soaked membrane (Fig. 3). To test the binding, lipophilic membranes were saturated with 10 mg/mL of THC, washed 10x with phosphate buffered saline (1% Tween-20), incubated with ScFv-EGFP, or ScFv-mNG, washed 3x with phosphate buffered saline (1% Tween-20), and imaged in Azure Biosystems 600. The excitation and emission wavelengths were 395 nm, and 509 nm, respectively.

![](https://static.igem.org/mediawiki/parts/e/ec/T--Queens_Canada--SpotAssay.jpg)

Figure 3. THC membrane assay using purified ScFv-EGFP and ScFv-mNG. Membranes were saturated with 10 mg/mL of THC, washed, and probed with the purified ScFv. Only ScFv-mNG gave a positive signal in the assay

![](https://static.igem.org/mediawiki/2019/f/f7/T--Queens_Canada--Home_footer.png)

[![](https://static.igem.org/mediawiki/2019/b/b6/T--Queens_Canada--QGEM_name.png)](http://queensigem.ca/)

Queen's Genetically Engineered Machine Team

Contact us at: qgem@engsoc.queensu.ca

Follow us on:

[![](https://static.igem.org/mediawiki/2019/9/98/T--Queens_Canada--Facebook_logo.png)](https://www.facebook.com/iGEMQueens)
[![](https://static.igem.org/mediawiki/2019/e/e7/T--Queens_Canada--Instagram_logo.png)](https://www.instagram.com/queens_igem/)

[![](https://static.igem.org/mediawiki/2019/9/98/T--Queens_Canada--Twitter_logo.png)](https://twitter.com/igemqueens)