iGEM 2019 — Team British_Columbia

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

# Team:British Columbia/Notebook

Team:British\_Columbia - 2019.igem.org

[![](https://static.igem.org/mediawiki/2019/e/e0/T--british_columbia--paralyte_logo.png)](https://2019.igem.org/Team%3ABritish_Columbia)

### Table of Contents

* Notebook

* [PROJECT](https://2019.igem.org/Team%3ABritish_Columbia/Inspiration)

  + ![](https://static.igem.org/mediawiki/2019/d/d0/T--british_columbia--menu_sub_bar.png)
  + [Inspiration](https://2019.igem.org/Team%3ABritish_Columbia/Inspiration)
  + [Description](https://2019.igem.org/Team%3ABritish_Columbia/Description)
  + [Approach](https://2019.igem.org/Team%3ABritish_Columbia/Approach)
  + [Hardware](https://2019.igem.org/Team%3ABritish_Columbia/Hardware)
  + [Results](https://2019.igem.org/Team%3ABritish_Columbia/Results)
  + [Parts](https://2019.igem.org/Team%3ABritish_Columbia/Parts)
  + [Future](https://2019.igem.org/Team%3ABritish_Columbia/Future)
* [LABORATORY](https://2019.igem.org/Team%3ABritish_Columbia/Experiments)

  + ![](https://static.igem.org/mediawiki/2019/d/d0/T--british_columbia--menu_sub_bar.png)
  + [Experimental Design](https://2019.igem.org/Team%3ABritish_Columbia/Experiments)
  + [Protocols](https://2019.igem.org/Team%3ABritish_Columbia/Protocol)
  + [Notebook](https://2019.igem.org/Team%3ABritish_Columbia/Notebook)
  + [Safety](https://2019.igem.org/Team%3ABritish_Columbia/Safety)
* [MODEL](https://2019.igem.org/Team%3ABritish_Columbia/Model)
* [HUMAN PRACTICES](https://2019.igem.org/Team%3ABritish_Columbia/Human_Practices)

  + ![](https://static.igem.org/mediawiki/2019/d/d0/T--british_columbia--menu_sub_bar.png)
  + [Integrated Human Practices](https://2019.igem.org/Team%3ABritish_Columbia/Human_Practices)
  + [Education/Outreach](https://2019.igem.org/Team%3ABritish_Columbia/Public_Outreach)
  + [Collaborations](https://2019.igem.org/Team%3ABritish_Columbia/Collaboration)
* [TEAM](https://2019.igem.org/Team%3ABritish_Columbia/Team)

  + ![](https://static.igem.org/mediawiki/2019/d/d0/T--british_columbia--menu_sub_bar.png)
  + [Team](https://2019.igem.org/Team%3ABritish_Columbia/Team)
  + [Attributions](https://2019.igem.org/Team%3ABritish_Columbia/Attribution)
  + [Sponsors](https://2019.igem.org/Team%3ABritish_Columbia/Sponsors)
* [MEDALS](https://2019.igem.org/Team%3ABritish_Columbia/Medals)

![](https://static.igem.org/mediawiki/2019/7/76/T--british_columbia--notebook.png)


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

# Team:British Columbia/Experiments

Team:British\_Columbia - 2019.igem.org

[![](https://static.igem.org/mediawiki/2019/e/e0/T--british_columbia--paralyte_logo.png)](https://2019.igem.org/Team%3ABritish_Columbia)

### Table of Contents

* [SIGEX](#SIGEX)
  + Screening Plasmid
  + Metagenomic Library
  + Electroporation
  + Cell Sorting
  + Efficacy Test
* [Screening The Pre-existing E.coli Promoter Library](#ecoli)

+ OD Growth Curve
+ Screening with Saxitoxin

* [PROJECT](https://2019.igem.org/Team%3ABritish_Columbia/Inspiration)

  + ![](https://static.igem.org/mediawiki/2019/d/d0/T--british_columbia--menu_sub_bar.png)
  + [Inspiration](https://2019.igem.org/Team%3ABritish_Columbia/Inspiration)
  + [Description](https://2019.igem.org/Team%3ABritish_Columbia/Description)
  + [Approach](https://2019.igem.org/Team%3ABritish_Columbia/Approach)
  + [Hardware](https://2019.igem.org/Team%3ABritish_Columbia/Hardware)
  + [Results](https://2019.igem.org/Team%3ABritish_Columbia/Results)
  + [Parts](https://2019.igem.org/Team%3ABritish_Columbia/Parts)
  + [Future](https://2019.igem.org/Team%3ABritish_Columbia/Future)
* [LABORATORY](https://2019.igem.org/Team%3ABritish_Columbia/Experiments)

  + ![](https://static.igem.org/mediawiki/2019/d/d0/T--british_columbia--menu_sub_bar.png)
  + [Experimental Design](https://2019.igem.org/Team%3ABritish_Columbia/Experiments)
  + [Protocols](https://2019.igem.org/Team%3ABritish_Columbia/Protocol)
  + [Notebook](https://2019.igem.org/Team%3ABritish_Columbia/Notebook)
  + [Safety](https://2019.igem.org/Team%3ABritish_Columbia/Safety)
* [MODEL](https://2019.igem.org/Team%3ABritish_Columbia/Model)
* [HUMAN PRACTICES](https://2019.igem.org/Team%3ABritish_Columbia/Human_Practices)

  + ![](https://static.igem.org/mediawiki/2019/d/d0/T--british_columbia--menu_sub_bar.png)
  + [Integrated Human Practices](https://2019.igem.org/Team%3ABritish_Columbia/Human_Practices)
  + [Education/Outreach](https://2019.igem.org/Team%3ABritish_Columbia/Public_Outreach)
  + [Collaborations](https://2019.igem.org/Team%3ABritish_Columbia/Collaboration)
* [TEAM](https://2019.igem.org/Team%3ABritish_Columbia/Team)

  + ![](https://static.igem.org/mediawiki/2019/d/d0/T--british_columbia--menu_sub_bar.png)
  + [Team](https://2019.igem.org/Team%3ABritish_Columbia/Team)
  + [Attributions](https://2019.igem.org/Team%3ABritish_Columbia/Attribution)
  + [Sponsors](https://2019.igem.org/Team%3ABritish_Columbia/Sponsors)
* [MEDALS](https://2019.igem.org/Team%3ABritish_Columbia/Medals)

### Substrate-Induced Gene Expression (SIGEX)

### Purpose

Screening metagenomic DNA extracted from organisms co-existing with harmful algal blooms for transcription factors that respond to saxitoxin.

### Justification

Little information about the transcription factors that respond to saxitoxin is found in the literature.

As saxitoxin (STX) effectively inhibits neurons, it is not harmful to prokaryotes and those organisms can survive in the presence of it, so it is possible that STX can regulate their genomic activities.

### General Procedure

#### Screening Vector Construction

Our vector was designed to yield the highest possible chance of inducing a measurable response from an inserted section of metagenomic DNA. Fluorescence was used as a means of quantifying the strength of a STX response due to the fluorescence activated cell sorting equipment available to us. Our vector was also designed to be able to report a fluorescence signal regardless of the way the blunt-end fixed insert was inserted by having fluorescence genes (RFP and GFP) on either side of the insert. Our full vector design can be seen:

![](https://static.igem.org/mediawiki/2019/4/40/T--british_columbia--vector.png)

[Figure1: pSB1C3 vector added with forward rbs+GFP and reverse rbs+GFP, and a NruI cut side in between. More about the vector can be seen on our [Parts page](https://2019.igem.org/Team%3ABritish_Columbia/Parts)]

The vector was also designed with a NRU1 cut site for insertion of the metagenomic fragments. After the vector was digested, it was treated with rSAP to dephosphorylate the 5’ and 3’ end to minimize self ligation.

#### Metagenomic DNA Extraction

Based on algal bloom data from the CFIA, metagenomic DNA from environments known to have contained high concentrations of STX at the time samples were taken were then prepared for insertion into the screening vector. The DNA was first sheared to sizes of around 6kb and then blunt-end fixed. 6kb fragments of metagenomic DNA were subsequently ligated into the screening vector.

#### Transformation and Colony Collection:

Vector-insert pairs were transformed into E.coli by electroporation to yield the highest possible amount of colonies. Colonies were then counted and pooled via scraping where they would be frozen down until cell sorting

#### Cell Sorting

The library of E.coli cells containing vector-insert pairs were sorted using a fluorescence activated cell sorter. A first uninduced sort was always done to remove and constitutively expressed cells. Cells were then induced with 2ppm STX, sorted again and then the light fraction was pooled and plated. Colonies were inoculated into 96 well plates and induced again. Using a plate reader, fluorescence was measured and the clones that showed the highest relative fluorescence were noted.

#### Efficacy Test

The clones that showed the most STX induced fluorescence were plated in 384-well plates in replicates of 3 and in varying STX concentrations. The data showed an increasing trend of fluorescence with an increase is STX concentration within the range of 0.4ppm - 2ppm. In light of these positive results, we have sent in these samples for sequencing and our team is waiting on the results. We plan to do further efficacy test of the STX-inducible clones to further characterize the biosensor as well as data analysis on the DNA sequences.

### Screening The Pre-existing E.coli Promoter Library

### Purpose

Screening the pre-existing library for finding a promoter that can respond to STX

### Justification

In previous studies, promoters that can interact with the substances that do not normally find in the E. coli habitat have been identified from the pre-existing E. coli promoter library

To increase our chance of finding a promoter that can interact with STX

### General Procedure

#### Library Preparation

A [pre-existing E.coli K12 promoter library](https://www.ncbi.nlm.nih.gov/pubmed/16862137)[1] was replicated from 384 master plates and subsequently transferred to 1536 well plates using a liquid handler. 1536 well plates were used in order to decrease the amount of STX used in the experiments as it is expensive. We could also be confident the experiment could be carried out accurately due to the high accuracy of the liquid handler. An LB media contain 2ppm STX was added to each well in order to induce any potential STX-inducible promoters. A concentration of 2ppm was chosen as we did not want to miss any potential hits that would not be sensitive enough to be detectable at lower levels such as the CFIA’s testing standards.

![](https://static.igem.org/mediawiki/2019/7/75/T--british_columbia--1536.jpg)

[Figure2: The white plates on the left contain the media (LB, LB+STX, LB+HCl (control)) that were loaded into the wells on the 1536 microwell plates. The black plates on the right are black bottom 1536 well-plates.]

#### Screening:

Screening was conducted by a robotic workstation that stored the 1536-well plates in an incubator and removed them once every hour to measure fluorescence over the course of approximately 24 hours.

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

# Team:British Columbia/Results

Team:British\_Columbia - 2019.igem.org

[![](https://static.igem.org/mediawiki/2019/e/e0/T--british_columbia--paralyte_logo.png)](https://2019.igem.org/Team%3ABritish_Columbia)

### Table of Contents

* [Pre-experiements](#pre)
* [Substrate-Induced Gene Expression (SIGEX)](#SIGEX)

* [PROJECT](https://2019.igem.org/Team%3ABritish_Columbia/Inspiration)

  + ![](https://static.igem.org/mediawiki/2019/d/d0/T--british_columbia--menu_sub_bar.png)
  + [Inspiration](https://2019.igem.org/Team%3ABritish_Columbia/Inspiration)
  + [Description](https://2019.igem.org/Team%3ABritish_Columbia/Description)
  + [Approach](https://2019.igem.org/Team%3ABritish_Columbia/Approach)
  + [Hardware](https://2019.igem.org/Team%3ABritish_Columbia/Hardware)
  + [Results](https://2019.igem.org/Team%3ABritish_Columbia/Results)
  + [Parts](https://2019.igem.org/Team%3ABritish_Columbia/Parts)
  + [Future](https://2019.igem.org/Team%3ABritish_Columbia/Future)
* [LABORATORY](https://2019.igem.org/Team%3ABritish_Columbia/Experiments)

  + ![](https://static.igem.org/mediawiki/2019/d/d0/T--british_columbia--menu_sub_bar.png)
  + [Experimental Design](https://2019.igem.org/Team%3ABritish_Columbia/Experiments)
  + [Protocols](https://2019.igem.org/Team%3ABritish_Columbia/Protocol)
  + [Notebook](https://2019.igem.org/Team%3ABritish_Columbia/Notebook)
  + [Safety](https://2019.igem.org/Team%3ABritish_Columbia/Safety)
* [MODEL](https://2019.igem.org/Team%3ABritish_Columbia/Model)
* [HUMAN PRACTICES](https://2019.igem.org/Team%3ABritish_Columbia/Human_Practices)

  + ![](https://static.igem.org/mediawiki/2019/d/d0/T--british_columbia--menu_sub_bar.png)
  + [Integrated Human Practices](https://2019.igem.org/Team%3ABritish_Columbia/Human_Practices)
  + [Education/Outreach](https://2019.igem.org/Team%3ABritish_Columbia/Public_Outreach)
  + [Collaborations](https://2019.igem.org/Team%3ABritish_Columbia/Collaboration)
* [TEAM](https://2019.igem.org/Team%3ABritish_Columbia/Team)

  + ![](https://static.igem.org/mediawiki/2019/d/d0/T--british_columbia--menu_sub_bar.png)
  + [Team](https://2019.igem.org/Team%3ABritish_Columbia/Team)
  + [Attributions](https://2019.igem.org/Team%3ABritish_Columbia/Attribution)
  + [Sponsors](https://2019.igem.org/Team%3ABritish_Columbia/Sponsors)
* [MEDALS](https://2019.igem.org/Team%3ABritish_Columbia/Medals)

### Pre-experiments

### OD growth curve

### Screening the Pre-existing E.coli Library

We screened around ~2300 different promoters from an E. coli promoter library. Using the Labcyte Echo, the dispensing and fluorescence readings were done mechanically, over 24 hours. Over 4 rounds of screening, we managed to narrow down the library to 7 leads. The analysis was done by selected outliers that were 3 adjusted standard deviations from the median. Looking at the most recent screen, each data point was adjusted to the control (GFPsample/GFPcontrol), and normalized to 1 OD600 ensure accuracy and representation. Each sample had 3 replicates, and, only one 1 out of the 7 leads (row 4, column 18) had a more consistent spread across the 3 replicates. Additional tests, such as more replicates and sequencing, needs to be done to fully ascertain whether these do have the potential to be made into a robust biosensor for saxitoxin.

We can conclude that most likely not cell’s intrinsic mechanisms acting on the saxitoxin.

#### Supplementary Data Analysis Files

[1][First screening plate 1](https://drive.google.com/file/d/1iQVl8_TdnHg5vzRpWTOo6tZ0ASizeKBm/view?usp=sharing)

[2][First screening plate 2](https://drive.google.com/file/d/1fhFeWPQo2BxJNtr5Sgl2mLRIbSBvHb3Z/view?usp=sharing)

[3][First screening plate 3](https://drive.google.com/file/d/1Z-LbJdueXFQVvneojJV1OmBjB6tB6lrO/view?usp=sharing)

[4][Second screening (all data)](https://docs.google.com/spreadsheets/d/1QmR0XwSs_N9c2oTlwZQoZkrHyCJNsFwfrDGzx44HnXY/edit?usp=sharing)

### SIGEX

### Transformation data

![](https://static.igem.org/mediawiki/2019/a/a4/T--british_columbia--transformation.jpg)
![](https://static.igem.org/mediawiki/2019/1/14/T--british_columbia--ligcontrol.jpg)

For SIGEX, we transformed around 100,000 different clones, ligating our vector plasmid with the prepped metagenomic library. Using vector ligation controls, we determined that there were negligible amount of self-ligation vectors.

#### Supplementary Transformation data

[1] The pictures of the plates of our transformation can be found [here](https://drive.google.com/drive/folders/1-OFighoR3CHj-Bgvz8r9IOiDHqRRzfbS)

### Cell Sorting

![](https://static.igem.org/mediawiki/2019/1/1c/T--british_columbia--cellsort1.png)

After pooling all our clones, we used the Chipbio cell sorter to sort out our cells. Alongside saxitoxin, we screened biphenyl and P-coumaric acid, two common effector molecules ([source](http://regulondb.ccg.unam.mx/index.jsp)) found in the Saanich inlet (where we acquired our DNA). These compounds were tested as a way to confirm the efficacy of our method. If known promoters were found, it would be another way to prove our experiments worked.

Initially sorter sorts through 1 million cells from the pooled DNA library, removing the constitutively fluorescing cells above a certain threshold. As seen in the image below, 93,958 cells were kept while the others were sorted out.

![](https://static.igem.org/mediawiki/2019/c/c6/T--british_columbia--cellsort2.png)

In the second round of sorting, the cells were incubated in their respective effector compound before being sorted. In the saxitoxin sort, 2,741 cells expressed relatively strong fluorescence and was kept for further testing.

### Efficacy Test

After sorting the cells, we plated the cells and inoculated 96 well plates with the colonies. When the cells were grown up, we transferred them into a 384 well plate with their respective compounds. A screen was performed, and out of the many candidates a total of 9 different clones were chosen for the final tests. The experiment we ran was a titration experiment, with 5 different concentrations, over a time of 16 hours. The results of the test is shown below in the figures. Overall, many of the samples indicate a favourable increasing trend of increasing GFP expression when STX concentration increases. The 0.05ppm STX is seen to not be a high enough concentration to induce any increased relative fluorescence, with most responses starting at 0.4 ppm over some time. Some samples, such as sample 3, have surprising aberrations that may be due to experimental results, but more testing will be needed to ascertain the causes.

#### Data Analysis

![](https://static.igem.org/mediawiki/2019/b/b3/T--british_columbia--efficacy1.PNG)
![](https://static.igem.org/mediawiki/2019/a/a9/T--british_columbia--efficacy2.PNG)
![](https://static.igem.org/mediawiki/2019/4/48/T--british_columbia--efficacy3.PNG)
![](https://static.igem.org/mediawiki/2019/9/92/T--british_columbia--efficacy4.PNG)

[Note:Bar graph: All raw data was first normalized to 1 OD600. The bar graph shows all the relative fluorescence of each sample. Sample 2 and sample 9 did not grow or fluoresce and therefore not shown here. Rejected samples, such as replicate 1 for 2.0ppm 2ppm for all samples, were removed for that same reason. The data here is selected for the best overall spread at a time point (This is not necessarily indicative of strongest fluorescence expression. It is just the time point where the overall trend is easiest to visualize.)]

[Note2:Line graph: The data points of each well were divided by their control to obtain their fold increase compared to the non-fluorescing control. As seen, the control is located at 1 at the x axis; anything below 1 has less relative fluorescence compared to the control, and anything above 1 has more. ]

Since there did not seem to be any information about saxitoxin’s effect on E. coli growth, we needed to run some experiments to confirm this for ourselves. We ran a HCl solution control as the saxitoxin itself was stored in 3 mM HCl. The results seen from the experiments showed that saxitoxin
itself did not affect the growth curves of the E. coli, matching the growth curves of the control.

#### Supplementary Data Used in Analysis

[1] [OD600nm and GFP fluorescence data](https://drive.google.com/file/d/1hUUD_ONyAI-CjWxVDHFBBk_8gMCNoAyz/view?usp=sharing)

[2] [Per\_Sample titration comparison by median](https://drive.google.com/file/d/1xYDXsWfuwZ2RE9dF2BcPsCb0YjJv3aAl/view?usp=sharing)

### Conclusion

From over 100,000 different clones and millions of cells sorted, we finally narrowed it down to a handful of potential candidates for our biosensor. The next steps will be to run additional tests on these candidates to fully confirm whether they have to potential to be made into biosensors. Currently we are sequencing the lead hits we tested out, which unfortunately will not be done before the wiki freeze. If we do confirm our hits, we can move on to optimizing these inducible promoters into transcription-based biosensors for downstream applications.