iGEM 2020 — Team Imperial_College

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# Team:Imperial College/Notebook

![](https://static.igem.org/mediawiki/2020/e/e0/T--Imperial_College--logo.png)

* [Home](https://2020.igem.org/Team%3AImperial_College)
* [Project](https://2020.igem.org/Team%3AImperial_College/Description)
* [Experimental Work](https://2020.igem.org/Team%3AImperial_College/Experimental_Work)
* [Human Practices](https://2020.igem.org/Team%3AImperial_College/Human_Practices)
* [People](https://2020.igem.org/Team%3AImperial_College/Attributions)
* [For Judges](https://2020.igem.org/Team%3AImperial_College/Awards)

# Notebook

## 13.10.20 Preparing Agar Plates and Media

##### NoteBook: [**N3.10.20 Preparing Agar Plates and Media.pdf**](https://static.igem.org/mediawiki/2020/f/f2/T--Imperial_College--13_10_20_Preparing_Agar_Plates_and_Media.pdf)

## 14.10.20: GoldenGate Assemblies (Manual)

##### notebook: [14.10.20: GoldenGate Assemblies (Manual).pdf](https://static.igem.org/mediawiki/2020/9/9a/T--Imperial_College--14.10.20_GoldenGate_Assemblies_%28Manual%29.pdf)

## 14.10.20 Mini-prep BASIC SEVA 36 Plasmids

##### Notebook: [14.10.20 Mini-prep BASIC SEVA 36 Plasmids.pdf](https://static.igem.org/mediawiki/2020/a/a2/T--Imperial_College--14.10.20_Mini-prep_BASIC_SEVA_36_Plasmids.pdf)

## **16.10.20 Preparation of Linkers**

##### **Notebook:**[**16.10.20 Preparation of Linkers.pdf**](https://static.igem.org/mediawiki/2020/9/93/T--Imperial_College--16.10.20_Preparation_of_Linkers.pdf)

## **19.10.20 BASIC Assemblies (Manual)**

##### **Notebook:**[**19.10.20 BASIC Assemblies (Manual).pdf**](https://static.igem.org/mediawiki/2020/9/95/T--Imperial_College--19.10.20_BASIC_Assemblies_%28Manual%29.pdf)

## **19.10.20 Dry-run of Opentrons Scripts (MoClo, BASIC)**

##### **Notebook:**[**19.10.20 Dry-run of Opentrons Scripts (MoClo, BASIC).pdf**](https://static.igem.org/mediawiki/2020/a/a4/T--Imperial_College--19.10.20_Dry-run_of_Opentrons_Scripts_%28MoClo%2C_BASIC%29.pdf)

## **21.10.20 BASIC Validation Assemblies (OT2)**

##### **Notebook:**[**21.10.20 BASIC VALIDATION ASSEMBLIES (OT2).pdf**](https://static.igem.org/mediawiki/2020/9/90/T--Imperial_College--21.10.20_BASIC_Validation_Assemblies_%28OT2%29.pdf)

##### [**21.10.20 DNA\_BOT\_instructions\_v1.0.0.pdf**](https://static.igem.org/mediawiki/2020/b/b3/T--Imperial_College--21.10.20_DNA_BOT_instructions_v1.0.0.pdf)

##### [**21.10.20 1\_clip.ot2,py**](https://static.igem.org/mediawiki/2020/0/04/T--Imperial_College--21.10.20_1_clip_ot2.txt)

##### [**21.10.20 1\_clip\_ot.txt**](https://static.igem.org/mediawiki/2020/c/c8/T--Imperial_College--21.10.20_1_clip_ot.txt)

##### [**21.10.20 2\_purification.ot2.py**](https://static.igem.org/mediawiki/2020/e/e9/T--Imperial_College--21.10.20_2_purification.ot2.txt)

##### [**21.10.20 2\_purification.txt**](https://static.igem.org/mediawiki/2020/5/55/T--Imperial_College--21.10.20_2_purification.txt)

##### [**21.10.20 3\_assembly.ot2.py**](https://static.igem.org/mediawiki/2020/5/5d/T--Imperial_College--21.10.20_3_assembly.ot2.txt)

##### [**21.10.20 3\_assembly.txt**](https://2020.igem.org/File%3AT--Imperial_College--21.10.20_3_assembly.txt)

##### [**21.10.20 basic\_validation.xml**](https://static.igem.org/mediawiki/2020/a/aa/T--Imperial_College--21.10.20_basic_validation.txt)

##### [**21.10.20 construct.csv**](https://static.igem.org/mediawiki/2020/6/65/T--Imperial_College--21.10.20_construct.txt)

##### [**21.10.20 construct\_clip\_run\_info.csv**](https://static.igem.org/mediawiki/2020/d/d4/T--Imperial_College--21.10.20_construct_clip_run_info.txt)

##### [**21.10.20 construct\_final\_assembly\_run\_info.csv**](https://static.igem.org/mediawiki/2020/4/46/T--Imperial_College--21.10.20_construct_final_assembly_run_info.txt)

##### [**21.10.20 construct\_wells.txt**](https://static.igem.org/mediawiki/2020/1/12/T--Imperial_College--21.10.20_construct_wells.txt)

##### [**21.10.20 part\_linker\_1**](https://static.igem.org/mediawiki/2020/6/6c/T--Imperial_College--21.10.20_part_linker_1.txt)

##### [**21.10.20 part\_linker\_2.csv**](https://static.igem.org/mediawiki/2020/3/3d/T--Imperial_College--21.10.20_part_linker_2.txt)

## **22.10.20 Mini-prep of BioBricks**

##### **Notebook:**[**22.10.20 Mini-prep of BioBricks.PDF**](https://static.igem.org/mediawiki/2020/0/03/T--Imperial_College--22.10.20_Mini-prep_of_BioBricks.pdf)

## **22.10.20 MoClo Validation Assemblies (OT2)**

##### **Notebook:**[**22.10.20 MoClo Validation Assemblies (OT2).pdf**](https://static.igem.org/mediawiki/2020/4/4b/T--Imperial_College--22.10.20_MoClo_Validation_Assemblies_%28OT2%29.pdf)

##### [**22.10.20 MoClo\_Transformation\_instructions.pdf**](https://static.igem.org/mediawiki/2020/c/c4/T--Imperial_College--22.10.20_MoClo_Transformation_instructions.pdf)

##### [**22.10.20 construct.CSV**](https://static.igem.org/mediawiki/2020/7/70/T--Imperial_College--22.10.20_construct.txt)

##### [**22.10.20 moclO.txt**](https://static.igem.org/mediawiki/2020/a/a1/T--Imperial_College--22.10.20_moclo.txt)

##### [**22.10.20 moclo\_assembly\_protocol.py**](https://static.igem.org/mediawiki/2020/2/2e/T--Imperial_College--22.10.20_moclo_assembly_protocol.txt)

##### [**22.10.20 moclo\_validation.xml**](https://static.igem.org/mediawiki/2020/f/f8/T--Imperial_College--22.10.20_moclo_validation.txt)

##### [**22.10.20 parts\_1.csv**](https://static.igem.org/mediawiki/2020/8/8e/T--Imperial_College--22.10.20_parts_1.txt)

## **23.10.20 BioBricks Validation Assembly (OT2)**

##### **NOTEBOOK:**[**BioBricks Validation Assembly (OT2).pdf**](https://static.igem.org/mediawiki/2020/d/d9/T--Imperial_College--23.10.20_BioBricks_Validation_Assembly_%28OT2%29.txt)

##### [**23.10.20 bb\_assembly\_protocol (1).py**](https://static.igem.org/mediawiki/2020/3/36/T--Imperial_College--23.10.20_bb_assembly_protocol_%281%29.txt)

##### [23.10.20\_bb\_assembly\_protocol.py](https://static.igem.org/mediawiki/2020/e/ee/T--Imperial_College--23.10.20_bb_assembly_protocol.txt)

##### [23.10.20\_bb\_assembly\_protocol.txt](https://static.igem.org/mediawiki/2020/4/44/T--Imperial_College--23.10.20_bb_assembly_protocoltext.txt)

##### [**23.10.20 bb\_metainformation 2**](https://static.igem.org/mediawiki/2020/7/71/T--Imperial_College--23.10.20_bb_metainformation_2.txt)

##### [**23.10.20 bb\_metainformation**](https://static.igem.org/mediawiki/2020/3/3e/T--Imperial_College--23.10.20_bb_metainformation.txt)

##### [**23.10.20 bb\_validation\_actual\_level1.xml**](https://static.igem.org/mediawiki/2020/3/36/T--Imperial_College--23.10.20_bb_validation_actual_level1.txt)

##### [**23.10.20 bb\_validation\_actual\_level2.xml**](https://static.igem.org/mediawiki/2020/a/a0/T--Imperial_College--23.10.20_bb_validation_actual_level2.txt)

##### [**23.10.20 construct 2.csv**](https://static.igem.org/mediawiki/2020/4/4b/T--Imperial_College--23.10.20_construct_2.csv)

##### [**23.10.20 construct .csv**](https://static.igem.org/mediawiki/2020/0/01/T--Imperial_College--23.10.20_construct.csv)

##### [**23.10.20 parts\_1 2.csv**](https://static.igem.org/mediawiki/2020/5/5c/T--Imperial_College--23.10.20_parts_1_2.csv)

##### [**23.10.20 parts\_1.csv**](https://static.igem.org/mediawiki/2020/3/37/T--Imperial_College--23.10.20_parts_1.csv)

## **26.10.20 BASIC Validation Assemblies 2 (OT2)**

##### **Notebook:** [**BASIC Validation Assemblies 2 (OT2).pdf**](https://static.igem.org/mediawiki/2020/b/b0/T--Imperial_College--26.10.20_1_clip.ot2.txt)

##### [**26.10.20 1\_clip.ot2,py**](https://static.igem.org/mediawiki/2020/0/04/T--Imperial_College--21.10.20_1_clip_ot2.txt)

##### [**26.10.20 1\_clip\_ot.txt**](https://static.igem.org/mediawiki/2020/c/c8/T--Imperial_College--21.10.20_1_clip_ot.txt)

##### [**26.10.20 2\_purification.ot2.py**](https://static.igem.org/mediawiki/2020/e/e9/T--Imperial_College--21.10.20_2_purification.ot2.txt)

##### [**26.10.20 2\_purification.txt**](https://static.igem.org/mediawiki/2020/5/55/T--Imperial_College--21.10.20_2_purification.txt)

##### [**26.10.20 3\_assembly.ot2.py**](https://static.igem.org/mediawiki/2020/5/5d/T--Imperial_College--21.10.20_3_assembly.ot2.txt)

##### [**26.10.20 3\_assembly.txt**](https://2020.igem.org/File%3AT--Imperial_College--21.10.20_3_assembly.txt)

##### [**26.10.20 basic\_validation.xml**](https://static.igem.org/mediawiki/2020/a/aa/T--Imperial_College--21.10.20_basic_validation.txt)

##### [**26.10.20 construct.csv**](https://static.igem.org/mediawiki/2020/6/65/T--Imperial_College--21.10.20_construct.txt)

##### [**26.10.20 construct\_clip\_run\_info.csv**](https://static.igem.org/mediawiki/2020/d/d4/T--Imperial_College--21.10.20_construct_clip_run_info.txt)

##### [**26.10.20 construct\_final\_assembly\_run\_info.csv**](https://static.igem.org/mediawiki/2020/4/46/T--Imperial_College--21.10.20_construct_final_assembly_run_info.txt)

##### [**26.10.20 construct\_wells.txt**](https://static.igem.org/mediawiki/2020/1/12/T--Imperial_College--21.10.20_construct_wells.txt)

##### [**26.10.20 part\_linker\_1**](https://static.igem.org/mediawiki/2020/6/6c/T--Imperial_College--21.10.20_part_linker_1.txt)

##### [**26.10.20 part\_linker\_2.csv**](https://static.igem.org/mediawiki/2020/3/3d/T--Imperial_College--21.10.20_part_linker_2.txt)

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

# Team:Imperial College/Engineering

![](https://static.igem.org/mediawiki/2020/e/e0/T--Imperial_College--logo.png)

* [Home](https://2020.igem.org/Team%3AImperial_College)
* [Project](https://2020.igem.org/Team%3AImperial_College/Description)
* [Experimental Work](https://2020.igem.org/Team%3AImperial_College/Experimental_Work)
* [Human Practices](https://2020.igem.org/Team%3AImperial_College/Human_Practices)
* [People](https://2020.igem.org/Team%3AImperial_College/Attributions)
* [For Judges](https://2020.igem.org/Team%3AImperial_College/Awards)

**Tryptophan (Trp) Optimization**

Our intention after validation…

![](data:image/svg+xml;base64...)

if more lab time had been possible…

was to further proof SOAP lab’s usefulness…

By applying it to...

**A metabolic engineering problem!**

Our initial and more ambitious project involved using BASIC assembly in yeast for the first time to build a high tryptophan yielding *Saccharomyces cerevisiae* strain. In the past, work on Trp optimisation in
yeast was done in the groups of Michael Jensen and J. Keasling, who took a predictive engineering approach using machine learning (Jie Zhang et al., 2019). This would offer a sustainable alternative to chemical
synthesis. L-Tryptophan is an essential amino acid widely used in medicine, food and animal feed and has an annual demand above 50000 tons (Liuet at., 2019). From the onset, we wanted to optimise the pathway with
help of automation, as we realised that even our reduced design space would require over 200 assemblies, which was further inspiration for SOAP Lab.

1. Jie Zhang, Søren D. Petersen, Tijana Radivojevic, Andrés Ramirez, Andrés Pérez, Eduardo Abeliuk, Benjamín J. Sánchez, Zachary Costello, Yu Chen, Mike Fero, Hector Garcia Martin, Jens Nielsen, Jay D. Keasling & Michael
K. Jensen 2019, "Predictive engineering and optimization of tryptophan metabolism in 2 yeast through a combination of mechanistic and machine learning 3 models", *BioRxiv*.
2. Liu, Bilal, Luo, Zhao &
Iqbal 2019, "Metabolic Engineering and Fermentation Process Strategies for L-Tryptophan Production by Escherichia coli", *Processes,*vol. 7, no. 4, pp. 213.

**STEP 1**

We had planned to achieve our aim through the simultaneous knockdown of competing pathway enzymes and overexpression of important enzymes in the Trp biosynthesis pathway or the shikimate pathway.

We initially found potential targets through investigating the literature and the biosynthesis pathways and  complemented it with **flux balance analysis (FBA)**.

Whilst we were unable to carry out the lab work due to the Covid pandemic, we present this modelling and the experimental design on this page of our Wiki.

# Modelling

Head over to our [Modelling](https://2020.igem.org/Team%3AImperial_College/Model) page to see how we did the modelling for this project!

**Next Steps**

![](data:image/png;base64...)

Sadly, due to Covid-19 all the subsequent steps will     have to be performed after the competition

###### Future work

**·   ·   ·**

Our software is particularly useful in high-throughput experiments, such as combinatorial approaches that explore a large design space.

![](data:image/png;base64...)

The enzymes **Trp2** and **Aro4**catalyze the initial step of Trp biosynthesis and the first
step in aromatic amino acid biosynthesis respectively *(Saccharomyces cerevisiae* database).

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They will be **overexpressed**in their mutated form to abolish any feedback related inhibition and will be **placed downstream 6 different strength promoters**, with the aim of finding their
optimal expression levels.

![](data:image/png;base64...)

Overexpression of these enzymes is supported by (Jie Zhang et at., 2019).

Simultaneously, we will express a single sgRNA targeting either **Aro7, Aro8** or **CDC19**to achieve its downregulation. Three different sgRNAs will be tested for the three different genes.

![](data:image/png;base64...)

The sgRNA will bind a constitutively expressed and genome integrated dCas9 in Rodrigo Ledesma’s *S. cerevisiae*validated strain.

Upon binding, these will travel to the promoter region of the above-mentioned genes.

Impeding the binding of DNA polymerase and
thus down regulating gene expression.

Aro7 initiates the branch for tyrosine/phenylalanine biosynthesis, a competing pathway for Trp production, and Aro 8 is involved in Trp degradation. CDC19 codes for pyruvate kinase which feeds into the TCA cycle which also
diverts flux from Trp production *(Saccharomyces cerevisiae* database).

Thus, we believe downregulating these genes would lead to an increase in Trp production. This is further supported by flux balance analysis we conducted on
previous work.

The objective was to use SOAP-Lab to build a large combinatorial library of varying CRISPR-dCas9 based knockdowns of Aro8, Aro7, and CDC19 combined with varied strengths of overexpressions of Aro4 K229L and Trp2 S65R,S76L.
For the successful implementation of this project it required the design of BASIC parts for yeast including: 7 yeast promoters; a yeast terminator; the two genes to be overexpressed; nine different gRNAs; and finally parts
for the development of a yeast backbone.These parts have all been synthesised with thanks to Twist and IDT, and we hope to continue with this project in the near future.

![](data:image/png;base64...)

The illustrated construct/s will be integrated into a backbone, which will include an origin of replication, chloramphenicol resistance, Ura 3 homology sites for genomic integration, mScarlett (red fluorescence) and an Ura
3 marker gene. The references for the mutated enzymes shown in the construct are: Graf, R., Mehmann, B., and Braus, G.H. (1993). Analysis of feedback-resistant anthranilate 945 synthases from Saccharomyces cerevisiae. J.
Bacteriol. *175*, 1061–1068. – trp2; Hartmann, M., Schneider, T.R., Pfeil, A., Heinrich, G., Lipscomb, W.N., and Braus, G.H. (2003). 956 Evolution of feedback-inhibited / barrel isoenzymes by gene duplication and
a single mutation. 957 Proc. Natl. Acad. Sci. *100*, 862–867.

If the single gRNA experiments had been successful, we would have then proceeded with experiments involving an array of multiple gRNAs. In order for each gRNA in the array to be cleaved, they would be placed in the following
manner:  HammerHead Ribozyme — gRNA — HDV ribozyme and repeat (He et al., 2017). This would allow to direct multiple gRNA to the promoter sequence of a target gene simultaneously. This is desirable as it would allow
to test for a range of knockdowns, hopefully leading to an optimal knockdown percentage of a competing pathway. We thought this would be interesting especially in Trp optimisation, because competing pathways in this case
lead to the production of other amino acids, essential for cell survival. As there is a trade off between cell survival and Trp production in the presence of competing pathway knockdown, we intended to find the optimal
knockdown percentage between 0 and 100%.

He, Y., Zhang, T., Yang, N., Xu, M., Yan, L., Wang, L., Wang, R. & Zhao, Y. 2017, "Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing", *Journal of genetics and genomics,*vol.
44, no. 9, pp. 469-472.

## Screening and gene expression analysis

### 1. Violacein pathway

To allow for screening, we will insert the violacein pathway as a visual output, which uses Trp as a precursor. An increase of Trp should lead to an increase of violacein production.

### 2. Liquid Chromatography / Mass Spectrometry (LCMS)

This initial screen would then be confirmed through LC/MS.

### 3. Real time quantitative PCR (RT-qPCR) and sequencing

Finally, gene up and down regulations will be measured through RT-qPCR and correct plasmid construction will be confirmed through sequencing.

We believe this part of the project to be exciting hope for two reasons –   firstly  we will demonstrate the application of BASIC to yeast for the first time, and  secondly we hope to achieve significant improvements
to Trp yield. If  unsuccessful, we will still proof the usefulness of the software in high-throughput experiments.

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

# Team:Imperial College/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:Imperial College/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)