iGEM 2020 — Team Manchester

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# Team:Manchester/Notebook

# 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:Manchester/Engineering

[![NavBarLogo](https://static.igem.org/mediawiki/2020/archive/1/1b/20201021180227%21T--Manchester--NavBarLogo.png)](https://2020.igem.org/Team%3AManchester)

[Overview](https://2020.igem.org/Team%3AManchester)

Project

[Description](https://2020.igem.org/Team%3AManchester/Description)
[Medals](https://2020.igem.org/Team%3AManchester/Medals)
[Project Values](https://2020.igem.org/Team%3AManchester/Project_Values)
[Safety](https://2020.igem.org/Team%3AManchester/Safety)
[Engineering success](https://2020.igem.org/Team%3AManchester/Engineering)
[Results and Future Steps](https://2020.igem.org/Team%3AManchester/Proof_Of_Concept)
[Implementation](https://2020.igem.org/Team%3AManchester/Implementation)
[Wiki Study](https://2020.igem.org/Team%3AManchester/Wiki_Study)

Human Practices

[Introduction](https://2020.igem.org/Team%3AManchester/Human_Practices)
[Surveys](https://2020.igem.org/Team%3AManchester/Surveys)
[Interviews](https://2020.igem.org/Team%3AManchester/Interviews)
[YouTube Analysis](https://2020.igem.org/Team%3AManchester/Media_Analysis)
[Responsibility and Reflections](https://2020.igem.org/Team%3AManchester/Responsibility_Reflections)

Modelling

[Model](https://2020.igem.org/Team%3AManchester/Model)
[Pathway Design](https://2020.igem.org/Team%3AManchester/Pathway_Design)
[Flux Balance Analysis](https://2020.igem.org/Team%3AManchester/Modelling)
[Chemical Analysis](https://2020.igem.org/Team%3AManchester/Chemical_Analysis)

Entrepreneurship

[Introduction](https://2020.igem.org/Team%3AManchester/Entrepreneurship)
[Business Report](https://2020.igem.org/Team%3AManchester/Business_Report)
[Business Plan](https://2020.igem.org/Team%3AManchester/Business_Plan)
[Learning Resource](https://2020.igem.org/Team%3AManchester/Contribution)

Team

[Our Team](https://2020.igem.org/Team%3AManchester/Team)
[Partnership](https://2020.igem.org/Team%3AManchester/Partnership)
[Collaborations](https://2020.igem.org/Team%3AManchester/Collaborations)
[Journal](https://2020.igem.org/Team%3AManchester/Journal)
[Attributions](https://2020.igem.org/Team%3AManchester/Attributions)

[Judging Form](https://igem.org/2020_Judging_Form?id=3340)
☰

# Engineering success

* We modified the experimental design of our project to allow us to work effectively during this unusual iGEM year.
* We successfully mapped our project to the Design–Build–Test–Learn (DBTL) cycle of synthetic biology.
* We demonstrate engineering success throughout the project.
* We apply the DBTL philosophy to each of the areas of the project individually.

#### Engineering success is and has always been a key component of both iGEM and Synthetic Biology. In the approximation to engineering Synthetic Biology adopted the Design–Build–Test–Learn (DBTL) cycle of the traditional engineering disciplines, and iGEM projects are generally carried out in accordance to this concept. This year, the COVID-19 pandemic has disrupted the traditional view of the cycle, and has forced many teams, including ours, to reconsider their approach.

#### Our measure for engineering success has been the application of the aforementioned cycle both at a whole-project scale, and individually to all the different aspects of our project

#### The DBTL cycle can be found throughout our project in our approach to almost every page and activity, some notable examples are:

* [Surveys](https://2020.igem.org/Team%3AManchester/Surveys), [Wiki Study](https://2020.igem.org/Team%3AManchester/Wiki_Study) and [Media Analysis](https://2020.igem.org/Team%3AManchester/Media_Analysis) from our [Human Practices](https://2020.igem.org/Team%3AManchester/Human_Practices) section.
* [Pathway Design](https://2020.igem.org/Team%3AManchester/Pathway_Design) and [Flux Balance Analysis](https://2020.igem.org/Team%3AManchester/Modelling) from [Modelling](https://2020.igem.org/Team%3AManchester/Model) section.
* [Results](https://2020.igem.org/Team%3AManchester/Engineering).
* [Business plan](https://2020.igem.org/Team%3AManchester/Business_Plan) and [Business report](https://2020.igem.org/Team%3AManchester/Business_Report) from our [Entrepreneurship](https://2020.igem.org/Team%3AManchester/Entrepreneurship) section.

![Image 1](https://static.igem.org/mediawiki/2020/thumb/5/5b/T--Manchester--flowchart_full.png/1599px-T--Manchester--flowchart_full.png)

## DESIGN

#### The DESIGN platform is for the modelling and design of pathways, and DNA constructs; it includes several iterations for the optimization of the process.

## Our DESIGN platform

![Flowchart 2](https://static.igem.org/mediawiki/2020/d/d4/T--Manchester--eng_suc2.png)

#### Our approach to design this year was divided in the following parts:

#### **[Pathway Design](https://2020.igem.org/Team%3AManchester/Pathway_Design)**: We accomplished a full pathway design by combining computational retrosynthetic and assembly methods with chemical analysis.

#### **[Enzyme Selection](https://2020.igem.org/Team%3AManchester/Pathway_Design)**: We used a self-hosted version of [Selenzyme](https://doi.org/10.1093/bioinformatics/bty065), an online enzyme selection to find enzymes to catalyze the reactions in our designed pathway.

#### **[Modelling](https://2020.igem.org/Team%3AManchester/Model)**: Informs the design itself, and it is used to further characterize the designed pathway as well as validate it. In addition it helps with design for the

#### **DNA Parts design**: When designing our DNA parts, we had to consider and optimise each of the following variables to produce a viable plasmid that would successfully work in our selected chassis.

* Codon optimisation.
* RBS design.
* Promoter design.
* Assembly method.
* Chassis selection.

#### We designed two parts for *Escherichia coli* strain DH5alpha. These two parts were among the Top 10 enzymes proposed by [Selenzyme](https://2020.igem.org/Team%3AManchester/Pathway_Design): two variants 4-hydroxyphenylpyruvate dioxygenase one from *Pseudomonas aeruginosa* (paHPPD), which can be seen Figure 3, and another one from *Streptomyces avermitilis* (saHPPD), which can be seen in Figure 4.

![Figure 3](https://static.igem.org/mediawiki/2020/3/3f/T--Manchester--eng_suc3.png)

###### **Figure 3. paHPPD construct, IPTG regulated.**

![Figure 4](https://static.igem.org/mediawiki/2020/9/99/T--Manchester--eng_suc4.png)

###### **Figure 4. saHPPD construct, IPTG regulated.**

## BUILD

#### The BUILD platform from the DBTL cycle is responsible for the production of parts for the subsequent assembly and transformation of the chassis. There is a growing trend for automation in this platform of the cycle.

![Flowchart 3](https://static.igem.org/mediawiki/2020/thumb/0/08/T--Manchester--eng_suc5.png/587px-T--Manchester--eng_suc5.png)

#### The Build section for us this year is quite simple, as we are constructing single assembly, single gene parts. The process involves the following steps:

* Commercial DNA synthesis
* PCR for part preparation
* Setup for pathway assembly
* Purification for quality checking
* Transformation of chassis

#### Plans for the future include assembling complex parts for overproduction of Tyrosine, as well as overexpression of HPPD to increase the yield of the chassis.

## TEST

#### The TEST platform of the “Design-Build-Test-Learn Cycle” is for tracking and assaying the products emanating from the BUILD platform. This process involves using different functional targeted analytical chemistry techniques and untargeted metabolomics to determine how the technology can be improved. In this sense the TEST platform acts as a debugging tool for the rest of the cycle. The results from the TEST platform feed back into the cycle to inform researchers how to extend both the laboratory scale and scale-up and scale-down processes.

![Flowchart 4](https://static.igem.org/mediawiki/2020/thumb/d/d8/T--Manchester--eng_2uc_6.png/956px-T--Manchester--eng_2uc_6.png)

#### After chassis transformation with our designed plasmids if the characteristic red colouration of Hipposudoric Acid appears, we will grow *E. coli* on a standard growth media and begin our product extraction and purification.

#### We have a choice to extract either the precursor HGA or our desired product Hipposudoric Acid. HGA is a known phenolic acid and can be easily identified by mass spectrometry (MS). The results of a MS analysis of HGA is a molecular weight of 168mu and relevant fragments at 66, 94, 122 and 150 mu (1). This is what we would expect to see, if this was not the case it implies there is a mistake within the DESIGN and BUILD platforms and we would have to go back to modify these earlier stages.

#### In contrast Hipposudoric Acid is a relatively uncharacterised compound and there is currently no MS standard. Hashimoto *et al.* (2007) attempted to characterise Hipposudoric Acid using 1HNMR, UV and mass spectral data however, due to the unstable nature of the acid it had to first be converted into a more stable derivative. This was achieved through a reduction of the pigment and subsequent methylation and silylation to produce a stable derivative that could be crystallised from methanol (2). It is likely that next year's team would have to follow this protocol in order to analyse Hipposudoric Acid. It has been deduced that the molecular weight of Hipposudoric Acid is 328.3, this is the first test that shall be performed to confirm that we have achieved our desired compound (2).

#### According to [Royal Society Publishing](https://royalsocietypublishing.org/rsos/chemical-compounds) when characterising a new organic compound one must “provide unequivocal support for the purity and assigned structure of all compounds” and this is achieved through a range of different analytical techniques. These are the protocols that will be performed in the laboratory next year.

#### **Analytical Analysis:**

* Elemental analysis within +/-0.4% of the calculated value is required to confirm 95% sample purity and corroborate isomeric purity.
* NMR spectra (1H, 13C).
* HPLC traces - determination of enantiomeric excess of non-racemic, chiral substances.
* Retention times.
* Mosher Ester/Chiral Shift Reagent analysis.
* Gel electrophoresis.

#### **Physical:**

* Boiling/melting point.
* Specific rotation.
* Refractive index - compared to known literature.
* Crystalline compounds - document method of recrystallisation.

#### **Spectroscopic:**

* Mass spectra.
* Infrared spectra - support functional group modifications.
* Diagnostic assignments.

#### Hipposudoric Acid is a small molecule that can theoretically [polymerise indefinitely](https://2020.igem.org/Team%3AManchester/Chemical_Analysis) producing a long chain; this means that the NMR data should be tabulated. Furthermore, it is possible that not all of these protocols will be relevant; however, because of the novelty of Hipposudoric Acid, we cannot determine at this stage which protocols are useful for characterisation of our compound.

#### After performing these experiments the data received for HGA and Hipposudoric Acid will be processed and analysed in preparation for the LEARN platform of the Design-Build-Test-Learn Cycle.

## LEARN

#### ![Flowchart 5](https://static.igem.org/mediawiki/2020/8/8e/T--Manchester--eng_suc7.jpg)

#### In the final stage of the cycle the results collected in the TEST platform are used to determine if any changes are needed in the DESIGN and BUILD platforms; the LEARN platform identifies how the project can be improved. Mechanistic modeling and artificial intelligence approaches such as machine learning can be applied to extract value from data sets. Learning is used to generate testable hypotheses that are incorporated into the next cycle resulting in a gradual development and improvement of our biotechnology over time. Once the process is optimised the final section of our project involves the moving from lab scale experiments towards commercial manufacturing, this is called Scale-Up. This process is usually accompanied by its own DBTL Cycle and involves up-scaling the fermentation capacity including further bioengineering to suit the bioprocess. This stage may induce unexpected costs and to align with our project values we may have to adopt our method accordingly to reduce retail prices.

## References

#### Literature

###### 1. Cabras, P., Angioni, A., Tuberoso, C., Floris, I., Reniero, F., Guillou, C., Ghelli, S., (1999) Homogentisic Acid: A Phenolic Acid as a Marker of Strawberry-Tree (Arbutus unedo) Honey, Journal of Agricultural and Food Chemistry, 47, 4064-4067

###### 2. Hashimoto, K., Saikawa, Y., Nakata, M., (2009) Studies on the red sweat of the Hippopotamus amphibius, Pure and Applied Chemistry, 79, 507-517

#### Bibliography

###### The Royal Society Publishing, Characterising new chemical compounds & measuring results, 2020, Available at: https://royalsocietypublishing.org/rsos/chemical-compounds Accessed: 20/09/2020

###### ACS Publications, Guidelines for Characterisation of Organic Compounds, 2020, Available at: https://pubs.acs.org/page/jacsat/submission/org\_character.html Accessed: 20/09/2020

###### Christopher Jonson,The Bio-Engineering Cycle, (N/A) Available at: https://biofoundries.org/design-build-test-learn Accessed: 09/10/2020

###### SYNBIOCHEM, SYNBIOCHEM pipeline, (N/A) Available at: http://synbiochem.co.uk/synbiochem-pipeline/ Accessed: 09/10/202

#### Figures

###### Flow chart made by Madeleine Webster-Harris 2020 in Lucidchart.com

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 *igem2020manchester@gmail.com*

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# Team:Manchester/Experiments

# 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:Manchester/Results

[![NavBarLogo](https://static.igem.org/mediawiki/2020/archive/1/1b/20201021180227%21T--Manchester--NavBarLogo.png)](https://2020.igem.org/Team%3AManchester)

[Overview](https://2020.igem.org/Team%3AManchester)

Project

[Description](https://2020.igem.org/Team%3AManchester/Description)
[Medals](https://2020.igem.org/Team%3AManchester/Medals)
[Project Values](https://2020.igem.org/Team%3AManchester/Project_Values)
[Safety](https://2020.igem.org/Team%3AManchester/Safety)
[Engineering success](https://2020.igem.org/Team%3AManchester/Engineering)
[Results and Future Steps](https://2020.igem.org/Team%3AManchester/Proof_Of_Concept)
[Implementation](https://2020.igem.org/Team%3AManchester/Implementation)
[Wiki Study](https://2020.igem.org/Team%3AManchester/Wiki_Study)

Human Practices

[Introduction](https://2020.igem.org/Team%3AManchester/Human_Practices)
[Surveys](https://2020.igem.org/Team%3AManchester/Surveys)
[Interviews](https://2020.igem.org/Team%3AManchester/Interviews)
[YouTube Analysis](https://2020.igem.org/Team%3AManchester/Media_Analysis)
[Responsibility and Reflections](https://2020.igem.org/Team%3AManchester/Responsibility_Reflections)

Modelling

[Model](https://2020.igem.org/Team%3AManchester/Model)
[Pathway Design](https://2020.igem.org/Team%3AManchester/Pathway_Design)
[Flux Balance Analysis](https://2020.igem.org/Team%3AManchester/Modelling)
[Chemical Analysis](https://2020.igem.org/Team%3AManchester/Chemical_Analysis)

Entrepreneurship

[Introduction](https://2020.igem.org/Team%3AManchester/Entrepreneurship)
[Business Report](https://2020.igem.org/Team%3AManchester/Business_Report)
[Business Plan](https://2020.igem.org/Team%3AManchester/Business_Plan)
[Learning Resource](https://2020.igem.org/Team%3AManchester/Contribution)

Team

[Our Team](https://2020.igem.org/Team%3AManchester/Team)
[Partnership](https://2020.igem.org/Team%3AManchester/Partnership)
[Collaborations](https://2020.igem.org/Team%3AManchester/Collaborations)
[Journal](https://2020.igem.org/Team%3AManchester/Journal)
[Attributions](https://2020.igem.org/Team%3AManchester/Attributions)

[Judging Form](https://igem.org/2020_Judging_Form?id=3340)
☰

# Results and Future Steps

* With **external help** we expressed two HPPD enzymes for the production of HGA.
* We compared the yield of the two recombinant strains.
* We validated the results through Mass Spectrometry.
* We outlined future steps for next year’s team.

####

## COVID Impact

#### Due to the COVID-19 pandemic and the lockdown and distancing measures imposed as a result in the UK, we have been unable to work in the laboratory ourselves. As discussed above this has made it difficult to follow the DBTL model as we would have done under different circumstances.

#### In order to gain some laboratory results in the face of this challenging situation we looked for alternative ways to carry out the experimental work.

#### We first attempted to perform the laboratory work ourselves with Dr Daniel Schindler, our supervisor, in his laboratory at the Max Planck Institute of Terrestrial Microbiology, in Marburg, Germany. However, this was impossible due to time constraints and the potential need to quarantine upon arrival in Germany and upon return to the UK.

#### We have been lucky in that our host institution, the [Manchester Institute of Biotechnology](http://www.mib.manchester.ac.uk/) of the [University of Manchester](https://www.manchester.ac.uk/), and some of its members have been able to continue carrying out laboratory work during the pandemic and thus we were able to collaborate to produce the following results.

## The Solution

#### We have worked closely with Dr Kris Nino Valdehuesa to design our constructs, and with Dr Mark Dunstan and Dr Andrew Curin from SYNBIOCHEM, the [Synthetic Biology Research Centre for Fine and Speciality Chemicals](http://synbiochem.co.uk/) in the Manchester Institute of Biotechnology, who built and expressed them for us.

#### As mentioned, due to the COVID-19 lockdown measures in place between March and July, our ability to carry out experimental research in the laboratory had to be outsourced to our collaborators at MIB. Aditionally, we were under a time-constraint due to it being July by the time the laboratory reopened and even later when our experiments were able to be started. As a result of these constraints we had to narrow down our list of desired experiments and thus prioritised the production and validation of HGA, our [predicted precursor](https://2020.igem.org/Team%3AManchester/Pathway_Design/) of Hipposudoric Acid.

#### The enzymes cloned and expressed are two variants of 4-Hydroxyphenylpyruvate dioxygenase chosen from the output from our [Pathway Design](https://2020.igem.org/Team%3AManchester/Pathway_Design/) enzyme selection tool, Selenzyme. One of them is the variant from *Pseudomonas aeruginosa* (paHPPD) and the other the variant from *Streptomyces averimilits* (saHPPD).

#### The chassis used for expression was *Escherichia coli* strain DH5alpha grown in the following growth conditions. Overnight seed cultures were grown from freshly transformed colonies in the desired production media at 37 °C with shaking at 180 rpm. Seed cultures were inoculated 1/50 into fresh media (1.2 mL in 96-deepwell blocks with breathable seals) and grown at 30 °C, 950 rpm shaking, 75% humidity. When cultures reached OD600 = 1.5 they were induced with the addition of 100 µM IPTG, 3 mM substrate (tyrosine) and Ascorbic acid, as appropriate, then transferred back to the 30 °C shaker-incubator for 24 h. Cultures were processed for mass spectrometry analysis using a Hamilton Star robotic platform.

* Lysogeny broth or Terrific Phosphate Broth phosphate buffered(TBP)
* 0.4 % Glycerol
* Kanamycin
* IPTG in the induced cultures

#### **Table 1. Production of HGA in uninduced and induced cultures.**

#### ![](https://static.igem.org/mediawiki/2020/5/5d/T--Manchester--Results_1.png)

#### As seen in **Table 1**, the induced recombinant DH5alpha with the saHPPD gene produced the highest yields of HGA, when grown in the TBP media. The validation of the chemical identity of the product was achieved through Mass Spectrometry by comparing the compound produced by the bacteria with a standard, as seen in **Figure 1**.

#### ![Figure 1](https://static.igem.org/mediawiki/2020/2/25/T--Manchester--Results_2.png)

###### **Figure 1. Mass spectrometry data for the three replicates from the DH5alpha saHPPD recombinant strain, and the standard of HGA.**

#### Samples were analysed on a triple quadrupole tandem mass spectrometer (Xevo TQ-S; Waters MS Technologies) connected to an Acquity Ultra Performance Liquid Chromatography system (Acquity UPLC; H-Class, Waters). HGA was stabilised by addition of ascorbic acid (reducing agent), but if the culture is left exposed to oxygen it starts turning red at 24h, and progresses through various shades of red to brown in the following days and weeks as seen on **Figure 2**.

#### ![Figure 2](https://static.igem.org/mediawiki/2020/thumb/5/55/T--Manchester--Results_3.png/1599px-T--Manchester--Results_3.png.jpeg)

###### **Figure 2. Picture of plated cultures highlighting oxygen sensitivity of HGA. Colonies were grown at 37˚ for 24hrs and then incubated for 2 weeks at 4˚ degrees.**

## Future Steps

#### The experimental results outlined on this page and our [modelling](https://2020.igem.org/Team%3AManchester/Model) work come together to support our hypothesis that Homogentisic Acid is a promising precursor compound for the production of Hipposudoric Acid.

#### This year’s special circumstances have forced us to reduce our experimental assays, and have pushed us to become a Two Phase Project. The experiments presented here were not carried out by our team members, but are due to the generous help of the researchers at our host institution. The following steps are clear:

* Expressing other HPPD enzymes, and HPPD analogues found through our modelling to find the most efficient ones.
* Purifying Hipposudoric Acid.
* Characterizing the basic chemical properties of the compound.
* Testing UV protective and anti-microbial properties of the produced compound.

#### Furthermore , a UV filter is only one component of a sunscreen, great consideration must go into choosing all the additional ingredients that will make up the sunscreen matrix included in the final product. Thorough testing must be carried out to ensure that the excipients do not undermine the UV-filtering effect as well as being non-toxic to aquatic life.

#### Additionally, sunscreens need to undergo several rounds of testing to be in accordance with both European and UK Laws and Regulations, and next year’s team will explore the necessary experimental protocols.

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 *igem2020manchester@gmail.com*

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