iGEM 2020 — Team CLS_CLSG_UK

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CLS CLSG UK/Notebook

[![](https://static.igem.org/mediawiki/2020/9/96/T--CLS_CLSG_UK--full_logo_without_background.png)](https://2020.igem.org/Team%3ACLS_CLSG_UK)

Team

[People](https://2020.igem.org/Team%3ACLS_CLSG_UK/People)

[Attributions](https://2020.igem.org/Team%3ACLS_CLSG_UK/Attributions)

[Inclusivity](https://2020.igem.org/Team%3ACLS_CLSG_UK/Inclusion)

[Contribution](https://2020.igem.org/Team%3ACLS_CLSG_UK/Contribution)

Human Practices

[Human Practices](https://2020.igem.org/Team%3ACLS_CLSG_UK/Human_Practices)

[Implementation](https://2020.igem.org/Team%3ACLS_CLSG_UK/Implementation)

[Collaborations](https://2020.igem.org/Team%3ACLS_CLSG_UK/Collaborations)

[Communication](https://2020.igem.org/Team%3ACLS_CLSG_UK/Education)

[Integrated Human Practices](https://2020.igem.org/Team%3ACLS_CLSG_UK/Integrated_Human_Practices)

[Handbook Quizzes](https://2020.igem.org/Team%3ACLS_CLSG_UK/Handbook_Quizzes)

Hardware

[Background](https://2020.igem.org/Team%3ACLS_CLSG_UK/Hardware)

[Design](https://2020.igem.org/Team%3ACLS_CLSG_UK/Hardware_Design)

[Cost analysis](https://2020.igem.org/Team%3ACLS_CLSG_UK/Cost_analysis)

[Next steps](https://2020.igem.org/Team%3ACLS_CLSG_UK/Next_Steps)

Modelling

[Introduction](https://2020.igem.org/Team%3ACLS_CLSG_UK/Model)

[Cocaine diffusion](https://2020.igem.org/Team%3ACLS_CLSG_UK/Cocaine%20diffusion)

[Enzyme concentration](https://2020.igem.org/Team%3ACLS_CLSG_UK/Enzyme_concentration)

[Total Breakdown](https://2020.igem.org/Team%3ACLS_CLSG_UK/Total_Breakdown)

[Evaluation](https://2020.igem.org/Team%3ACLS_CLSG_UK/Evaluation)

Project

[Description](https://2020.igem.org/Team%3ACLS_CLSG_UK/Description)

[Design](https://2020.igem.org/Team%3ACLS_CLSG_UK/Design)

[Engineering](https://2020.igem.org/Team%3ACLS_CLSG_UK/Engineering)

[Parts](https://2020.igem.org/Team%3ACLS_CLSG_UK/Parts)

[Methods](https://2020.igem.org/Team%3ACLS_CLSG_UK/Methods)

[Results](https://2020.igem.org/Team%3ACLS_CLSG_UK/Results)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CLS CLSG UK/Engineering

[![](https://static.igem.org/mediawiki/2020/9/96/T--CLS_CLSG_UK--full_logo_without_background.png)](https://2020.igem.org/Team%3ACLS_CLSG_UK)

Team

[People](https://2020.igem.org/Team%3ACLS_CLSG_UK/People)

[Attributions](https://2020.igem.org/Team%3ACLS_CLSG_UK/Attributions)

[Inclusivity](https://2020.igem.org/Team%3ACLS_CLSG_UK/Inclusion)

[Contribution](https://2020.igem.org/Team%3ACLS_CLSG_UK/Contribution)

Human Practices

[Human Practices](https://2020.igem.org/Team%3ACLS_CLSG_UK/Human_Practices)

[Implementation](https://2020.igem.org/Team%3ACLS_CLSG_UK/Implementation)

[Collaborations](https://2020.igem.org/Team%3ACLS_CLSG_UK/Collaborations)

[Communication](https://2020.igem.org/Team%3ACLS_CLSG_UK/Education)

[Integrated Human Practices](https://2020.igem.org/Team%3ACLS_CLSG_UK/Integrated_Human_Practices)

[Handbook Quizzes](https://2020.igem.org/Team%3ACLS_CLSG_UK/Handbook_Quizzes)

Hardware

[Background](https://2020.igem.org/Team%3ACLS_CLSG_UK/Hardware)

[Design](https://2020.igem.org/Team%3ACLS_CLSG_UK/Hardware_Design)

[Cost analysis](https://2020.igem.org/Team%3ACLS_CLSG_UK/Cost_analysis)

[Next steps](https://2020.igem.org/Team%3ACLS_CLSG_UK/Next_Steps)

Modelling

[Introduction](https://2020.igem.org/Team%3ACLS_CLSG_UK/Model)

[Cocaine diffusion](https://2020.igem.org/Team%3ACLS_CLSG_UK/Cocaine%20diffusion)

[Enzyme concentration](https://2020.igem.org/Team%3ACLS_CLSG_UK/Enzyme_concentration)

[Total Breakdown](https://2020.igem.org/Team%3ACLS_CLSG_UK/Total_Breakdown)

[Evaluation](https://2020.igem.org/Team%3ACLS_CLSG_UK/Evaluation)

Project

[Description](https://2020.igem.org/Team%3ACLS_CLSG_UK/Description)

[Design](https://2020.igem.org/Team%3ACLS_CLSG_UK/Design)

[Engineering](https://2020.igem.org/Team%3ACLS_CLSG_UK/Engineering)

[Parts](https://2020.igem.org/Team%3ACLS_CLSG_UK/Parts)

[Methods](https://2020.igem.org/Team%3ACLS_CLSG_UK/Methods)

[Results](https://2020.igem.org/Team%3ACLS_CLSG_UK/Results)

[![](https://static.igem.org/mediawiki/2020/7/78/T--CLS_CLSG_UK--Project_Engineering_Banner.jpg)](https://static.igem.org/mediawiki/2020/7/78/T--CLS_CLSG_UK--Project_Engineering_Banner.jpg)

Engineering

##### Credit - Bill Oxford@unsplash

* [Research](#Subtitle1)
  [Design](#Subtitle2)
  [Build](#Subtitle3)
  [Test](#Subtitle4)

# Engineering

In order to achieve engineering success we have done a number of things: we have used and reused an iterative design model by which we were able to continuously improve our project. This led to us tackling and solving many of our projects problems using creative synthetic biology methods. As well as this, given we had very limited lab work, we were able to plan, in full, all of the lab work that we hoped to carry out, as well as plan and document the results obtained from our experiments.

Our iterative design process:

[![](https://static.igem.org/mediawiki/2020/f/fa/T--CLS_CLSG_UK--EngineeringPic.png)](https://static.igem.org/mediawiki/2020/f/fa/T--CLS_CLSG_UK--EngineeringPic.png)

Throughout our [Design](https://2020.igem.org/Team%3ACLS_CLSG_UK/Design) it is clear that our process was a largely iterative one. We often had to go back to the drawing board late on in the project after new information came to light. A few exemplar examples of this are described below.

We first researched heavily into enzymes to use to best break down the cocaine and then settled on the bacterial cocaine esterase from rhodococcus. We then designed multiple genetic circuits that wld be able to produce the enzyme but settled on a simple but effective constitutive production. We then went on to build a model, with help from Imperial's team and then used our model to test the efficiency of our enzymes. From this we learnt that if we were able to increase the thermal half life of our enzyme, the ability to break down cocaine would be drastically increased. Having learnt this new information we then delved back into a mass of papers to try and use our newly learnt information. We discovered that there is a mutant form of our original enzyme with an increased thermal stability. We then went back and incorporated this enzyme into our original design. We have since uploaded both parts to the registry for future teams to build, test and learn with.

More generally though our whole project followed this rough design cycle:

## Research

Our project began with much research into a variety of different fields and ideas. However, we soon realised that a project involving our local area would be most beneficial. Our eyes immediately turned towards the River Thames. While the River Thames is now considered one of the cleanest rivers in a major city, we knew there were still large issues. Some of our team members came across articles on the rising cocaine concentrations in the River Thames and the demise of the European Eel. This of course needed validation by scientific journals, but soon we realised that cocaine concentrations in the river Thames was having a large impact upon the European eel species. Please have a look at our [description](https://2020.igem.org/Team%3ACLS_CLSG_UK/Description) page for more about our research.

## Design

We designed our project with all of our research in mind, we wanted to implement synthetic biology in a safe, efficient and sustainable manner to try and address this problem. Our [design](https://2020.igem.org/Team%3ACLS_CLSG_UK/Design) page details exactly how we achieved this. Our design ended up with three novel genetic circuits. As well as this we designed a new hardware device that is capable of getting in-situ, quantitative data that can be used by organisations, governments and other iGEM teams.

## Build

We built various mathematical models that we could use to evaluate our system. As well as this, we built and adapted protocols so that, in the lab, we would be able to construct our circuits. Please see our [methods](https://2020.igem.org/Team%3ACLS_CLSG_UK/Methods) and page for further detail.

## Test and improve

We tested one of our constructs, the Synthetic Adhesin, the results of which can be seen [here](https://2020.igem.org/Team%3ACLS_CLSG_UK/Results). As well as this, we used our model to test the use of different enzymes within our system to see their effectiveness. This can be seen [here](https://2020.igem.org/Team%3ACLS_CLSG_UK/Model).

In addition, we planned out all our labwork, which included how we would approach and deal with problems. This can be seen [here](https://2020.igem.org/Team%3ACLS_CLSG_UK/Methods). While we were not able to complete all our labwork, we were able to test and show that one of our constructs works as expected, which is further detailed in our results.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CLS CLSG UK/Experiments

[![](https://static.igem.org/mediawiki/2020/9/96/T--CLS_CLSG_UK--full_logo_without_background.png)](https://2020.igem.org/Team%3ACLS_CLSG_UK)

Team

[People](https://2020.igem.org/Team%3ACLS_CLSG_UK/People)

[Attributions](https://2020.igem.org/Team%3ACLS_CLSG_UK/Attributions)

[Inclusivity](https://2020.igem.org/Team%3ACLS_CLSG_UK/Inclusion)

[Contribution](https://2020.igem.org/Team%3ACLS_CLSG_UK/Contribution)

Human Practices

[Human Practices](https://2020.igem.org/Team%3ACLS_CLSG_UK/Human_Practices)

[Implementation](https://2020.igem.org/Team%3ACLS_CLSG_UK/Implementation)

[Collaborations](https://2020.igem.org/Team%3ACLS_CLSG_UK/Collaborations)

[Communication](https://2020.igem.org/Team%3ACLS_CLSG_UK/Education)

[Integrated Human Practices](https://2020.igem.org/Team%3ACLS_CLSG_UK/Integrated_Human_Practices)

[Handbook Quizzes](https://2020.igem.org/Team%3ACLS_CLSG_UK/Handbook_Quizzes)

Hardware

[Background](https://2020.igem.org/Team%3ACLS_CLSG_UK/Hardware)

[Design](https://2020.igem.org/Team%3ACLS_CLSG_UK/Hardware_Design)

[Cost analysis](https://2020.igem.org/Team%3ACLS_CLSG_UK/Cost_analysis)

[Next steps](https://2020.igem.org/Team%3ACLS_CLSG_UK/Next_Steps)

Modelling

[Introduction](https://2020.igem.org/Team%3ACLS_CLSG_UK/Model)

[Cocaine diffusion](https://2020.igem.org/Team%3ACLS_CLSG_UK/Cocaine%20diffusion)

[Enzyme concentration](https://2020.igem.org/Team%3ACLS_CLSG_UK/Enzyme_concentration)

[Total Breakdown](https://2020.igem.org/Team%3ACLS_CLSG_UK/Total_Breakdown)

[Evaluation](https://2020.igem.org/Team%3ACLS_CLSG_UK/Evaluation)

Project

[Description](https://2020.igem.org/Team%3ACLS_CLSG_UK/Description)

[Design](https://2020.igem.org/Team%3ACLS_CLSG_UK/Design)

[Engineering](https://2020.igem.org/Team%3ACLS_CLSG_UK/Engineering)

[Parts](https://2020.igem.org/Team%3ACLS_CLSG_UK/Parts)

[Methods](https://2020.igem.org/Team%3ACLS_CLSG_UK/Methods)

[Results](https://2020.igem.org/Team%3ACLS_CLSG_UK/Results)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:CLS CLSG UK/Results

[![](https://static.igem.org/mediawiki/2020/9/96/T--CLS_CLSG_UK--full_logo_without_background.png)](https://2020.igem.org/Team%3ACLS_CLSG_UK)

Team

[People](https://2020.igem.org/Team%3ACLS_CLSG_UK/People)

[Attributions](https://2020.igem.org/Team%3ACLS_CLSG_UK/Attributions)

[Inclusivity](https://2020.igem.org/Team%3ACLS_CLSG_UK/Inclusion)

[Contribution](https://2020.igem.org/Team%3ACLS_CLSG_UK/Contribution)

Human Practices

[Human Practices](https://2020.igem.org/Team%3ACLS_CLSG_UK/Human_Practices)

[Implementation](https://2020.igem.org/Team%3ACLS_CLSG_UK/Implementation)

[Collaborations](https://2020.igem.org/Team%3ACLS_CLSG_UK/Collaborations)

[Communication](https://2020.igem.org/Team%3ACLS_CLSG_UK/Education)

[Integrated Human Practices](https://2020.igem.org/Team%3ACLS_CLSG_UK/Integrated_Human_Practices)

[Handbook Quizzes](https://2020.igem.org/Team%3ACLS_CLSG_UK/Handbook_Quizzes)

Hardware

[Background](https://2020.igem.org/Team%3ACLS_CLSG_UK/Hardware)

[Design](https://2020.igem.org/Team%3ACLS_CLSG_UK/Hardware_Design)

[Cost analysis](https://2020.igem.org/Team%3ACLS_CLSG_UK/Cost_analysis)

[Next steps](https://2020.igem.org/Team%3ACLS_CLSG_UK/Next_Steps)

Modelling

[Introduction](https://2020.igem.org/Team%3ACLS_CLSG_UK/Model)

[Cocaine diffusion](https://2020.igem.org/Team%3ACLS_CLSG_UK/Cocaine%20diffusion)

[Enzyme concentration](https://2020.igem.org/Team%3ACLS_CLSG_UK/Enzyme_concentration)

[Total Breakdown](https://2020.igem.org/Team%3ACLS_CLSG_UK/Total_Breakdown)

[Evaluation](https://2020.igem.org/Team%3ACLS_CLSG_UK/Evaluation)

Project

[Description](https://2020.igem.org/Team%3ACLS_CLSG_UK/Description)

[Design](https://2020.igem.org/Team%3ACLS_CLSG_UK/Design)

[Engineering](https://2020.igem.org/Team%3ACLS_CLSG_UK/Engineering)

[Parts](https://2020.igem.org/Team%3ACLS_CLSG_UK/Parts)

[Methods](https://2020.igem.org/Team%3ACLS_CLSG_UK/Methods)

[Results](https://2020.igem.org/Team%3ACLS_CLSG_UK/Results)

[![](https://static.igem.org/mediawiki/2020/5/53/T--CLS_CLSG_UK--Project_Results_Banner.jpg)](https://static.igem.org/mediawiki/2020/5/53/T--CLS_CLSG_UK--Project_Results_Banner.jpg)

Results

##### Credit - National Cancer Institute@unsplash

* [Overview](#Subtitle1)

# Results

## Overview

While we were very happy with the results that we achieved, given we thought that any lab work was going to be impossible, we still had a few issues. The primary issue being that until we came to actually examine the plates under the light microscope we did not realise that we were unable to focus on the bottoms of the wells at a high power (400x). Given our initial plans to obtain quantitative data was to take images of the wells and then run them through an image processing software such as photoshop, we could not now do that. And while we could see the stark difference in the different wells we needed a new way of displaying and potentially quantifying the results. So we decided to construct a key with which each well’s coverage could be ranked on 100x magnification. By doing this it would create a system to present the results in a clear and easily understandable way, but also get quantitative data, though the use of this data given its subjective nature is contentious.

Looking at the plate layout (include link):

[![](https://static.igem.org/mediawiki/2020/b/b1/T--CLS_CLSG_UK--Results1.png)](https://static.igem.org/mediawiki/2020/b/b1/T--CLS_CLSG_UK--Results1.png)

In order to make analysis of results easier we have divided the plate into quadrants:

[![](https://static.igem.org/mediawiki/2020/3/33/T--CLS_CLSG_UK--Result2.png)](https://static.igem.org/mediawiki/2020/3/33/T--CLS_CLSG_UK--Result2.png)

One would expect that the highest level of adherence would be located in wells 1A to 5D, and that within this area there would be a decreasing gradient in the level of adherence due to the increased washing times. As well as this you would expect nothing to be present in columns 6 and 7 and that if any bacteria was present it would be in low quantities. As for the effect that washing with the synthetic sewer water rather than PBS would have we were not sure as there were arguments that it would increase and decrease the adherence of our bacteria.

First, looking at the plate washed only with PBS:

[![](https://static.igem.org/mediawiki/2020/7/77/T--CLS_CLSG_UK--Results3.png)](https://static.igem.org/mediawiki/2020/7/77/T--CLS_CLSG_UK--Results3.png)

The first thing that is apparent, is that the increased washing has a significant effect on both strains of bacteria to adhere, as well as this column 6 and 7 remained empty. But outside of this we can also see that while there are areas where bacteria are able to adhere in very low amounts the level of adherence where both the SA E. coli and the GFP was present is clearly the highest. Comparing all four quadrants gives that the average density per well in quadrant 1 is 1.55, compared to 0.55 in quadrant 2 with 0.4 and 0.6 in quadrants 3 and 4 respectively.

Looking at the plate washed with our synthetic sewer water:

[![](https://static.igem.org/mediawiki/2020/d/dd/T--CLS_CLSG_UK--Results4.png)](https://static.igem.org/mediawiki/2020/d/dd/T--CLS_CLSG_UK--Results4.png)

The same key was used for both plates so that we can compare them. The plate showed a similar trend to the first one regarding the effect of increased washing, as well as that, columns 6 and 7 remained empty. Similar to plate 1 the highest density of bacteria is in quadrant 1, where we would expect them, and other areas all have a very low adherence rate. By calculating the average well density for each quadrant, which is calculated by dividing the sum of all the values given to each quadrant by twenty, a bar chart can be made.

[![](https://static.igem.org/mediawiki/2020/5/5e/T--CLS_CLSG_UK--Graph1.png)](https://static.igem.org/mediawiki/2020/5/5e/T--CLS_CLSG_UK--Graph1.png)

Displaying the results in this way clearly shows a number of things. Firstly, the synthetic sewer water had a significant effect on the ability of the E. coli to adhere, this can be seen through all the quadrants. In every quadrant the adherence in the synthetic sewer water is lower than that in the PBS by a sizable amount, this would indicate that the synthetic sewer water does have an effect on the ability of the bacteria to adhere to antigenic surfaces. We had a few theories as to why that is but we aren’t sure. Of course the presence of the ions caused this, but which ions specifically and how and why they caused this would require further experimentation. However, one conclusion that can be drawn that in sewer conditions the ability of the non-modified E. Coli to adhere was very close to zero, so the presence of the synthetic adhesin is very important to enable our bacteria to adhere to the surface.

The second thing that is apparent from this experiment is that no matter the conditions, the effect that the expression of the synthetic adhesin has on the adherence of the bacteria is large. This can be seen through the graphical representation of the results as well as the visual representation both of which are above. But we also wanted to see if we could show this mathematically and so ran an independent student's t-test. The two data sets used were the adherence of the S.A. E. Coli with GFP and the adherence of all of the other three conditions. This gives us:

[![](https://static.igem.org/mediawiki/2020/6/6c/T--CLS_CLSG_UK--Equation1.png)](https://static.igem.org/mediawiki/2020/6/6c/T--CLS_CLSG_UK--Equation1.png)

This value can be rounded to 2.45 and when compared with the degrees of freedom table this value shows that these two sets of data are statistically significant to a 98% certainty. Therefore proving that the synthetic adhesin has a significant effect on the ability of the E. Coli to adhere.

Of course we are also aware that one could call this assumption flawed because of the manner with which the data was collected. While this is true the strong correlation between the quantitative and qualitative data as well as the fact that or findings are in line with our hypothesis and with the findings of the research paper published on this we are therefore able to assume that the expression of a synthetic adhesin when in the presence of a complimentary antigenic surface significantly increases the ability of that bacteria to adhere to the surface.