iGEM 2020 — Team AFCM-Egypt

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# Team:AFCM-Egypt/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:AFCM-Egypt/Engineering

[* INTRODUCTION](#introduction)
[* DESIGNING OF PARTS](#designParts)
[* PARTS OPTIMIZATION](#partsOptimisation)
[* PROJECT IMPROVEMENTS](#projectImprovements)
[* Assembly of plasmids](#aop)
[* References](#literature)

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* [Home (current)](https://2020.igem.org/Team%3AAFCM-Egypt)
* Team

  [Team members](https://2020.igem.org/Team%3AAFCM-Egypt/team)
  [Attributions](https://2020.igem.org/Team%3AAFCM-Egypt/Attributions)
* Project

  [Description](https://2020.igem.org/Team%3AAFCM-Egypt/Description)
  [Engineering](https://2020.igem.org/Team%3AAFCM-Egypt/Engineering)
  [Contribution](https://2020.igem.org/Team%3AAFCM-Egypt/Contribution)
  [Implementation](https://2020.igem.org/Team%3AAFCM-Egypt/Implementation)
  [Entrepreneurship](https://2020.igem.org/Team%3AAFCM-Egypt/Entrepreneurship)
  [Modeling](https://2020.igem.org/Team%3AAFCM-Egypt/Model)
  [Design](https://2020.igem.org/Team%3AAFCM-Egypt/Design)
* Parts

  [Basic Parts](https://2020.igem.org/Team%3AAFCM-Egypt/Parts)
  [Composite Parts](https://2020.igem.org/Team%3AAFCM-Egypt/Parts#composite)
* [Software](https://2020.igem.org/Team%3AAFCM-Egypt/Software)
* Human Practices

  [Human Practices](https://2020.igem.org/Team%3AAFCM-Egypt/Human_Practices)
  [Education](https://2020.igem.org/Team%3AAFCM-Egypt/Education)
  [Collaboration](https://2020.igem.org/Team%3AAFCM-Egypt/Collaborations)
  [Partnership](https://2020.igem.org/Team%3AAFCM-Egypt/Partnership)

## Introduction

This year challenged us to think of new ways of engineering our project under heavy constraints.
In order to work on our engineering we decided to go by the book and follow the engineering
design cycle, so in our journey we went into many phases of trial & error. However, the way we
implemented the Design, Build, Test cycle was heavily impacted by our surrounding circumstances.

![](https://static.igem.org/mediawiki/2020/a/a3/T--AFCM-Egypt--EngineeringItearationsAll.png)
Figure. illustrates the iterations that our team went through in the
process of engineering our vector.

Nevertheless, we attempted to adapt to the new norm by thinking outside of the box and building
upon/designing tools and resources to aid us in the conceptualization of our project.

Throughout this season, we kept revisiting our design, consulting various experts, resources and
taking the results of our modeling and simulations into account. We went through multiple
iterations in order to finally reach a successful engineering of our project. In this page, we
depict the multiple iterations of our design as guided by our engineering efforts. To view our
finalized suggested design, please consult our design page.

Before we jump into details, i’d like to summarize all what you are going to see in the next
pages. This year we decided to use a replicon-based plasmid, and a replicon is a region of DNA
or RNA, that replicates from a single origin of replication. Which is an essential component in
multiple viruses such as alphaviruses like EEV & SFV to replicate their structural genes.

A replicon mainly consists of four non structural proteins (NSPs) that are the main part that
cause the replication.

![](https://static.igem.org/mediawiki/2020/0/04/T--AFCM-Egypt--diagram-engineering.png)

This year we decided to use replicons for their self amplification abilities & high expression
of a desired gene or construct.

First we are going to show you our initial design and ideas followed by improvements and
additions that were added to them later on after integrated practices meetings

Finally we are going to show what we are planning to assemble to get experimental data so we can
get the best efficacy possible out of our vaccine.

Now let’s Dive in.

## Designing of parts and Fragments

### Initial Design

We planned to create two vectors

1.The first vector was made as a modular design that represented our
circuit in which its main parts were

| Biobrick | Type | Role | Length |
| --- | --- | --- | --- |
| [BBa\_K3504000](https://www.google.com/url?q=http://parts.igem.org/Part:BBa_K3504000&sa=D&ust=1603466402390000&usg=AOvVaw1Vc6QH17DL70eS9qRyQDjd) | Coding | Nsp4 for replicon | 1605 |
| [BBa\_K3504001](https://www.google.com/url?q=http://parts.igem.org/Part:BBa_K3504001&sa=D&ust=1603466402392000&usg=AOvVaw2INZmQyU_DqeclDjKrGgY0) | Coding | Nsp4 for replicon | 2382 |
| [BBa\_K3504002](https://www.google.com/url?q=http://parts.igem.org/Part:BBa_K3504002&sa=D&ust=1603466402394000&usg=AOvVaw23N3kNlHkXWz3tRaButYz8) | Coding | Nsp4 for replicon | 1671 |
| [BBa\_K3504003](https://www.google.com/url?q=http://parts.igem.org/Part:BBa_K3504003&sa=D&ust=1603466402396000&usg=AOvVaw1h7d20qfIMbYVd1YtKLdVa) | Coding | Nsp4 for replicon | 1821 |
| [BBa\_K3504004](https://www.google.com/url?q=http://parts.igem.org/Part:BBa_K3504004&sa=D&ust=1603466402398000&usg=AOvVaw2CcIi2NKyeAmpRWJVlSVC_) | Regulatory | Subgenomic promoter | 30 |
| [BBa\_K3504005](https://www.google.com/url?q=http://parts.igem.org/Part:BBa_K3504005&sa=D&ust=1603466402400000&usg=AOvVaw0PP_liz9fwC7lUv7D7-0nE) | DNA | Malat-1Triplex | 132 |
| [BBa\_K3504006](https://www.google.com/url?q=http://parts.igem.org/Part:BBa_K3504006&sa=D&ust=1603466402401000&usg=AOvVaw0PmkZLQK_iBrIKHsQJc78P) | Regulatory | mi-RNA FF6 | 64 |

Table (1)
![](https://static.igem.org/mediawiki/2020/a/a1/T--AFCM-Egypt--designCircuit.png)
Figure 1. Initial circuit design
![](https://static.igem.org/mediawiki/2020/7/7d/T--AFCM-Egypt--designCircuit2.png)
Figure 2. Initial circuit SBOL design

### Replicon design & Optimizations:

The main component of our replicon-based plasmid is the 4 non structural proteins by which we can
attain self-amplification of a downstream construct which would highly increase the expression
of our vaccine using the parts (BBa\_K3504000,BBa\_K3504001,BBa\_K3504002,BBa\_K3504003). Which
allowed an increased expression which was proven from literature and when combined with the part
BBa\_K3504004 as a Subgenomic promoter demonstrated the following expression results.

![](https://static.igem.org/mediawiki/2020/6/65/T--AFCM-Egypt--designPartChart.png)
Figure.3 Number of positive strand RNA Before and after self replication

We also found that we can improve our EEV alphavirus replicon amplification ability by addition
of selective mutations to the non-structural proteins all of which are shown in Table(2) &
Figures (4-9) below.

| Mutation Region | Nucleotide Mutations | Amino acid Mutations |
| --- | --- | --- |
| Nsp2 | A2619G | G110G |
| Nsp2 | G4576C | G763R |
| Nsp3 | A4951G | K94E |
| Nsp3 | A5399G | S243G |
| Nsp3 | G5436T,G5584A | E255D,V305M |

Table(2)
![](https://static.igem.org/mediawiki/2020/a/a4/T--AFCM-Egypt--aminoPlots.png)

### Initial Switch design

We designed an on-switch that was designed to further increase expression, stability and provide
protection from silencing. This switch had an “OR” logic gate to work this on switch was
activated
by either one of two miRNA:

1. the first of was mir-FF6 which was chosen according to data provided from literature that
   showed that it had highest expression in usage on cases of an on switch demonstrated in
   the figure 10.
![](https://static.igem.org/mediawiki/2020/4/43/T--AFCM-Egypt--switchPlot.png)
Figure 10. miRNA Fold change between various miRNAs, Showing that all of them can act as
an OFF motif but only few can actually act as an ON motif.
2. The second of them we choose it to be a prognostic marker for TNBC, our chosen miRNA was
   mir-373 and we choose it after comparing it with multiple candidates as we found it to be
   highly upregulated in TNBC patients and for it’s high stability as it had a minimum free
   energy of -36.40 kcal/mol.

![](https://static.igem.org/mediawiki/2020/f/f8/T--AFCM-Egypt--marker.png)
Figure11.TNBC prognostic marker mir-373 RNA fold

And this switch contained the part BBa\_K3504005 (Malat 1 triple helix) as a stabilization
domain to replace the function of the Poly A tail.
Finally we used multiple terminator sequences to be used as a degradation domain.

### Current design & improvements

![](https://static.igem.org/mediawiki/2020/0/05/T--AFCM-Egypt--finaldesign.png)
Figure12. Final circuit Design
![](https://static.igem.org/mediawiki/2020/5/5d/T--AFCM-Egypt--finalSbol.png)
Figure13.Final Sbol Design for the circuit

## Parts optimization

### MiRNA Optimization & binding site interaction

#### Methodology

First of all, we obtained DNA sequences of miRNA templates used in pPRIME-based cloning from
supplementary information on nature website which are FF3, FF4, FF5 and FF6.And by using RNAfold
Webserver tool, RNA sequence of the miRNAs was obtained in addition to their folding and
modeling with the energy of the thermodynamic ensemble (Kcal/mol).

Consequently, we selected the best base-pair probabilities and by using another tool called RNA
reserve and complementary sequence generator, The basepair probabilities’ complementary sequence
was gained.

Eventually, The IntaRNA interaction tool was used to determine the stability and binding energy
of the miRNAs with the circuit.

#### Results

| miRNA | Sequence of miRNA | free energy of the thermodynamic ensemble | Best stability for RNA interaction | Sequence of binding site |
| --- | --- | --- | --- | --- |
| FF3 | UCGAGUGCUGUUGACAGUGAGCGCACGAUAUGGGCUGAAUACAAA UAGUGAAGCCACAGAUGUAUUUGUAUUCAGCCCAUAUCGUUUGCC UACUGCCUCGGAG | -50.98 kcal/mol | -6kcal/mol | UCGAGUGCUGUUGACAGUGAGCGCACGAUAUGGGCUGAA UACAAAUAGUGAAGCCACAGAUGUAUUUGUAUUCAGCCC AUAUCGUUUGCCUACUGCCUCGGAG |
| FF4 | UCGAGUGCUGUUGACAGUGAGCGCCGCUUGAAGUCUUUAA UUAAAUAGUGAACCACAGAUGUAUUUAAUUAAAGACUUCA AGCGGUGCCUACUGCCUCGGAG | -51.23 kcal/mol | -3.14 kcal/mol | AGCUCACGACAACUGUCACUCGCGGCGAACUUCAGAAAU UAAUUUAUCACUUCGGUGUCUACAUAAAUUAAUUUCUGA AGUUCGCCACGGAUACGGAGCCUC |
| FF5 | TCGAGTGCTGTTGACAGTGAGCGAAGCACTCTGATTTGA CAATTATAGTGAAGCCACAGATGTATAATTGTCAAATCA GAGTGCTTTGCCTACTGCCTCGGAG | -53.24 kcal/mol | -10.99kcal/mol | AGCACUCUGAUUUGACAAUUAU |
| FF5 | TCGAGTGCTGTTGACAGTGAGCGAACCAAAGAGAT TCCTCATAAATAGTGAAGCCACAGATGTATTTATG AGGAATCTCTTTGGTTTGCCTACTGCCTCGGAG | -53.34 kcal/mol | -3.92kcal/mol | CGCUUGGUUUCUCUAAGGAGUAUUUAU |

![](https://static.igem.org/mediawiki/2020/6/67/T--AFCM-Egypt--stability.png)
![](https://static.igem.org/mediawiki/2020/9/93/T--AFCM-Egypt--EngineeringItearations2.png)

Figure: This Design->Build->Test Cycle Illustrates the process of SGP
optimization & selection

### Cse promoter Optimization

#### Methodology

Using RNAfold web server, we could predict secondary structures of single stranded RNA or DNA
sequences and obtain the free energy of the thermodynamic ensemble. Not only those but also we
could obtain a graphical output for the inserted sequence.

We used 3 sequences for SGP with 3 different lengths. Each one of them has 6 expressions; 3
expressions are measured by mVenus and the other 3 are measured by mKatewe and could obtain the
energy for each one of them by using RNAfold web server.

Eventually, we correlated the expressions of the sequences with the stability and another time
with the length by using the Pearson Correlation Coefficient Calculator tool.

For instance, by using RNAfold web server to obtain the free energy of the thermodynamic
ensemble for SGP41 with sequence (CCTGAATGGACTACGACATAGTCTAGTCCGCCAAGGCCACC) it was

-13.82 kcal/mol. And the same for each SGP till we get all free energies. Then we correlate them
with their expression using the Pearson Correlation Coefficient Calculator tool and again with
the length

![](https://static.igem.org/mediawiki/2020/5/59/T--AFCM-Egypt--cseChart.png)
Figure (1)
![](https://static.igem.org/mediawiki/2020/5/58/T--AFCM-Egypt--cseRNAFold.png)
Figure (2) The RNAfold of the sequence which has the least energy which is -13.82 kcal/mol.

| Sequence | Length of sequence | Stability | Expression mVenus | Expression mKate |
| --- | --- | --- | --- | --- |
| CCTGAATGGACTACGACATAGTCTAGTCCGCCAAGGCCACC | 41 | -13.82 | 0.05 | 0.13 |
| CCTGAATGGACTACGACATAGCCACC | 26 | -3.18 | 0.07 | 0.18 |
| CCTGAATGGAGCCACC | 16 | -1.02 | 0.04 | 0.16 |
| CCTGAATGGACTACGACATAGTCTAGTCCGCCAAGGCCACC | 41 | -13.82 | 0.33 | 0.35 |
| CCTGAATGGACTACGACATAGCCACC | 26 | -3.18 | 0.04 | 0.55 |
| CCTGAATGGAGCCACC | 16 | -1.02 | 0.025 | 0.667 |
| CCTGAATGGACTACGACATAGTCTAGTCCGCCAAGGCCACC | 41 | -13.82 | 0.04 | 0.667 |
| CCTGAATGGACTACGACATAGCCACC | 26 | -3.18 | 0.04 | 0.69 |
| CCTGAATGGAGCCACC | 16 | -1.02 | 0.02 | 1.15 |
|  |  |  | R value for stability -0.1648 | R value for stability 0.3094 |
|  |  |  | R value for length 0.3159 | R value for length -0.3479 |

#### Results

The end result of the correlation coefficient between the stability and the expression by mVenus
was R value equals -0.1648 and between stability and expression by mKate was 0.3094. On the
other hand, the correlation coefficient between the length and the expression by mVenus was R
value equals 0.3159 and between length and expression by mKate was -0.3479.

## Project improvements based on Integrated practices

We have been so focused on the self-amplification property that we missed another important
feature as after our integrated practices meeting with Dr.Noreen Wauford co-author of a
promising platform for RNA regulation called PERSIST have pointed us to the fact that we need to
consider the recognition and attack of immunity against our vector and mining literature for
more ways to increase our circuits efficiency we came with the following decisions and
conclusions that furthermore increased the total efficiency of our replicon vaccine.

we decided to add two protective mechanisms the first of which was to protect our vaccine from
recognition and attack by Cytotoxic lymphocytes which could lead to the degradation of cells
expressing our vaccine, so we added glycine-alanine repeats which has been proven to prevent
this presented by the part BBa\_K3504007 (Gly-Ala repeats) as we found that it would help protect
our circuit from innate immunity attack.

![](https://static.igem.org/mediawiki/2020/b/bc/T--AFCM-Egypt--ProjectImprovementCharts.png)
Figure14.CTLs degradation rate after addition of Gly ala vs Before adding it
![](https://static.igem.org/mediawiki/2020/8/86/T--AFCM-Egypt--GlyAla.png)
Figure15.Gly-Ala repeats amino acid sequence

The second was designing an OFF-Switch based on a modular NOR gate which depends on two miRNAs
as its inputs, the first of them being mir-126, which would help protect our vector from being
recognized and attacked by innate immunity if transfected to antigen presenting cells like DCs
as these cells express high level of mir-126 which would activate the switch and stop the
replication in these cells while preserving it in other cells.

And the other input being synthetic microRNA FF4 which can be administered to stop the
replication and work as a safety switch.

But We choose this microRNA between multiple choices as it experimentally showed strong
downregulation when used as off switch and furthermore for its highly stable structure as seen
in figure 10

With their binding sites being The part BBa\_K3504008 (Mir-126 binding site) and the BBa\_K3504019
(FF4 Binding site).

![](https://static.igem.org/mediawiki/2020/6/63/T--AFCM-Egypt--EngineeringItearations1.png)

Figure: This Design->Build->Test Cycle Illustrates the Immune evasion steps through Integrated
practices.
![](https://static.igem.org/mediawiki/2020/3/3e/T--AFCM-Egypt--NorGate.png)

Finally we decided to remove the ON-switch as its ability to protect from silencing was replaced
by Gly-Ala repeats and the OFF-switch.

## Assembly of plasmids

WFirst we decided to assemble two variants of our vaccine, the first one to be made using Equine
Encephalitis Virus (EEV) Non Structural Protein replicon parts and the second using Semliki
Forest Virus (SFV) parts with minor differences in design.

We decided to use Golden Gate assembly cloning system in the assembly of EEV plasmids while using
Simple restriction-enzyme ligation system in SFV Plasmids.

We separated the circuit to 8 fragments

The first fragment contains Origin of replication and the AmpR and promoter.

![](https://static.igem.org/mediawiki/2020/e/e2/T--AFCM-Egypt--EngineeringampR.png)

The second fragment contains CMV promoter , FF4 binding site & mir126 binding site.

![](https://static.igem.org/mediawiki/2020/b/b7/T--AFCM-Egypt--Engineeringcmvff4.png)

The 3rd to 6th fragments contained NSP1,NSP2,NSP3,NSP4 in that order with every fragment
containing only one NSP.

![](https://static.igem.org/mediawiki/2020/8/81/T--AFCM-Egypt--EngineeringNsp1-4.png)

The 7th fragment contained SubGenomic promoter, B-defensin, Badri and most importantly the chosen
epitopes to our vaccine.

![](https://static.igem.org/mediawiki/2020/5/52/T--AFCM-Egypt--EngineeringSGP30Gly.png)

The 8th and final fragment contained reporter genes and enhanced p2a+gsg And when designing our
reporter gene, we found that we could enhance its function by adding p2a cleavage site
separating the vaccine from the reporter gfp. And even further more increase its cleavage
ability by adding 3xflag & v5tag upstream of it.

![](https://static.igem.org/mediawiki/2020/b/b8/T--AFCM-Egypt--Engineeringv5Tag.png)

In order to choose between multiple design variations and select the best and most efficient one
we assembled multiple plasmid designs to experimentally & computationally compare between them
due to the lack of lab availability due to Covid-19 pandemic.

The factors we had to compare between were :-

1. Different versions of Multi-Epitope vaccine
2. Subgenomic promoter optimization
3. Off switch variants & the efficacy of mir-126

![](https://static.igem.org/mediawiki/2020/e/ee/T--AFCM-Egypt--EngineeringItearations3.png)

Figure: This Design->Build->Test Cycle Illustrates the process of alphavirus type selection

### Multi-epitope vaccine optimization:

First we are planning to design multiple versions of our vaccines to validate the best form of
the vaccine to be used as personalized immunization techniques according to using pSFV3 plasmid
assembled with different versions of the vaccine using in them SGP15 as the Subgenomic promoter.

![](https://2020.igem.org/File:T--AFCM-Egypt--ImplementationPSFV3.png)

Multi-epitope Vaccine V1

![](https://static.igem.org/mediawiki/2020/0/0d/T--AFCM-Egypt--ImplementationPSFV3%2BSGP.png)

Multi-epitope vaccine v2

![](https://static.igem.org/mediawiki/2020/8/82/T--AFCM-Egypt--ImplementationPSFV3-SGP.png)

Multi-epitope vaccine v3

### Nor Gate testing & Mir126 characterization

In efforts to characterize mir126 effects we are planning to assemble two plasmids one with the
mir126 binding site and one without it to experimentally show the effectiveness it adds to the
circuit.

![](https://static.igem.org/mediawiki/2020/6/69/T--AFCM-Egypt--ImplementationPEEV-SGP.png)

![](https://static.igem.org/mediawiki/2020/8/83/T--AFCM-Egypt--ImplementationPEEV-SGP2.png)

### Sgp Optimization

Finally we are planning to assemble 2 plasmids to experimentally decide which subgenomic
promoter would show higher expression abilities in which would be identified using reporter
genes.

![](https://static.igem.org/mediawiki/2020/6/69/T--AFCM-Egypt--ImplementationPEEV-SGP.png)

![](https://static.igem.org/mediawiki/2020/6/6d/T--AFCM-Egypt--ImplementationPEEV-SGP4.png)

However using mathematical modelling and simulation techniques we found out that SGP15 had
higher expression values which was confirmed from literature presented in Figure16 to be
confirmed after experimental testing .

![](https://static.igem.org/mediawiki/2020/2/25/T--AFCM-Egypt--ImplementationFigure16.png)

Figure 16.Sgp 30 expression vs SGP15

## References

1. DiAndreth, B., Wauford, N., Hu, E., Palacios, S., & Weiss, R. (2019). PERSIST: A
   programmable RNA regulation platform using CRISPR endoRNases. bioRxiv.
2. Wagner, T. E., Becraft, J. R., Bodner, K., Teague, B., Zhang, X., Woo, A., ... &
   Sanders, N. N. (2018). Small-molecule-based regulation of RNA-delivered circuits in
   mammalian cells. Nature chemical biology, 14(11), 1043-1050.
3. Hofacker, I. L. (2003). Vienna RNA secondary structure server. Nucleic acids research,
   31(13), 3429-3431.

### SPONSORS

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![](https://static.igem.org/mediawiki/2020/5/55/T--AFCM-Egypt--LOGO-BEGIN.png)

#### We Engineer To Clear..

@

igem.afcm@gmail.com

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# Team:AFCM-Egypt/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:AFCM-Egypt/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)