iGEM 2022 — Team paris-bettencourt

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

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| Protocols & Notebook |

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## Protocols

* *Bacillus Subtilis* Transformation [{DF}](https://static.igem.wiki/teams/4216/wiki/wiki/protocols/bacillus-subtilis-transformation.pdf)
* Conoly PCR for *E. coli* [PDF](https://static.igem.wiki/teams/4216/wiki/wiki/protocols/COLONY_PCR_E.COLI.pdf)
* Chemical transformation for *E. coli* [PDF](https://static.igem.wiki/teams/4216/wiki/wiki/protocols/E._Coli_CHEMICAL_TRANSFORMATION_.pdf)
* *E. coli* Heat-shock transformation [PDF](https://static.igem.wiki/teams/4216/wiki/wiki/protocols/E.coli_Transformation.pdf)
* Golden Gate Assembly using the Echo liquid handler
* Making electrocompetent *E. coli*\* cells [PDF](https://static.igem.wiki/teams/4216/wiki/wiki/protocols/ELECTROCOMPETENT_CELLS_.pdf)
* Gel Electrophoresis [PDF](https://static.igem.wiki/teams/4216/wiki/wiki/protocolsGel_Electrophoresis.pdf)
* Gibson Assembly [PDF](https://static.igem.wiki/teams/4216/wiki/wiki/protocolsGIBSON_ASSEMBLY_.pdf)
* Glycerol stocks [PDF](https://static.igem.wiki/teams/4216/wiki/wiki/protocolsGLYCEROL_STOCK.pdf)
* Golden Gate Assembly [PDF](https://static.igem.wiki/teams/4216/wiki/wiki/protocolsGOLDEN_GATE_ASSEMBLY__.pdf)
* Home-made *E. coli* competent cells with CaCl2. [PDF](https://static.igem.wiki/teams/4216/wiki/wiki/protocolsHome_Made_Competent_E.Coli_.pdf)
* Sequencing [PDF](https://static.igem.wiki/teams/4216/wiki/wiki/protocols/SEQUENCING_.pdf)
* *E. coli* transformation with TSS [PDF](https://static.igem.wiki/teams/4216/wiki/wiki/protocols/tss-transformation.pdf)

## Lab Notebook

### July 15, 2022 :

- Transformation pETDuet- p15

### Jul 17, 2022 :

- Liquid cultures of pACYCDuet-1 p17/pCDFDuet-1 p16/pCOLADuet-1 p18

### Jul 18, 2022 :

*- Antibiotic Resistance Assay*
*- Miniprep : pACYCDuet-1 p17/pCDFDuet-1 p16/pCOLADuet-1 p18*
*- Transformation dpB.015, dpB016, ppB.030*
*- Tranformation pETDuet-p15*

### Jul 19, 2022:

- Miniprep : pACYCDuet-1 p17/pCDFDuet-1 p16/pCOLADuet-1 p18
- Transformation :ppB.030/ppb.029 ; ppB.028/ppB.029 ; ppB.028/29/30
- Transformation: ppB.030/ppB.0.28

### Jul 20, 2022:

- Transformation of p15 in commercial comp cells
- Liquid culture all pDuet( exept pET) + GFP antoine + Moclo DVA et DVE

### Jul 26, 2022:

- CIDAR MoClo Vector UPGRADE
- IO rodeo potentiostat, electrode wiring
- Cloning PnasA for B.Subtilis

### Jul 28, 2022:

- Cloning Lvl1

### Aug 1, 2022:

- Miniprep: dpB 004, 006, 010, 013, 016, 075
- NanoDrop of minipreps
- First test of potentiostat (success!)
- Cultures on kanamycin plates

### Aug 2, 2022 :

- UV view on cultures
- Miniprep (NEW PROTOCOL): dpB 006, pp038
- NanoDrop miniprep dpB006, pp038
- Cloning of dp.012
- Colony PCR + gel v35

### Aug 3, 2022 :

- Glycerol stock
- Colony PCR v35 + gel

### Aug 4, 2022 :

- Transformation dpB.008 dpB.009 dpB.011
- Miniprep by new protocol 1st part, nanodrop, liquid culture
- Colony PCR

### Aug 7, 2022 :

- Miniprep by new protocol 2nd part + results analysis

### Aug 9, 2022:

- Sequencing dpB.003,dpB.005,dpB.006,dpB.007,dpB.008, dpB 009, dpB.011, dpB.012, dpB.013

### Aug 10, 2022:

- Cloning for dpB 018 & 019 (Act106 & Inv106) from JL
- Miniprep dpB.001, dpB.002, dpB.006, dpB.010, dpB.014

### Aug 12, 2022:

- Miniprep dpB 018 & 019 (Act 106 & Inv 106)

### Aug 14,2022:

- Miniprep dpB 018 & 019 (Act 106 & Inv 106)

### Aug 15, 2022:

- Processor parts building and characterisation
- Test of diff.concentration of inducers for dpB.010

### Aug 16, 2022:

- Testing Microscope slides for Hardware

### Aug 17, 2022:

- Simple OD Meter
- Miniprep Moclo parts for Echo (pp.048, pp.049,pp.050)

### Aug 18, 2022:

- Upgade pDuet with V35, Vector cloning
- Primer dilution and sequencing prep

### Aug 19, 2022:

- Plate Reader Inducers Marionettes

### Aug 21, 2022:

- Golden Gate pDuet and G Blocks
- Bacteria Electroshock

### Aug 22, 2022:

- Plate Reader Assay Inv & Act
- Transformation of b.sub
- Perform lacZ screening assay
- Perfom starch test
- Characterisation of the promotor
- Building all pDuet with the g.block and try different combinaison
to test the processor and the marionettes system with multiple plasmids

### Aug 23, 2022:

- building of all dpB in the 3 pDUET by goldengate
- Plate Reader Assay pOxyS
- Get the electroshock 96 well plate as well as the microscope slide ready for experiments
- Ideally get one experiment on Amplitude running
- Get the Uri Alon Library working for experiments (prepare batch culture and clone the library)

### Aug 24, 2022:

- Transformation of all pDUET in comp cells

### Aug 25, 2022:

- Plate Reader Assay pSoxR

### Aug 26,2022:

- Plate Reader assay pOxyS - Higher concentrations

### Aug 27, 2022:

- Microscope electroshock
- Subtitoolkit Replica Plate
- Electrical conductivity meter - 555 Timer

### Aug 28,2022 :

- Gibson Assembly Debug
- Sequencing pDuets

### Aug 29, 2022:

- Microscope electroshock
- Uri Alon Library

### Sep 1, 2022:

- Lac Z colony PCR

### Sep 2, 2022:

- Output Conductivity Characterization #1

### Sep 8, 2022:

- [FACS] Electroshocking bacteria on 96 Well plate

### Sep 10, 2022

- Miniprep for Paris\_Inv Gibson
- Growth Curve Uri Alon m9

### Sep 11, 2022

- PCR for Paris\_Inv

### Sep 13, 2022:

- PCR for Paris\_Inv Q5

### Sep 15, 2022:

- Transformation Partnership Training Constructs

### Sep 18, 2022:

- Streak of Paris\_Inv and Training constructs
- Gibson Assembly Paris\_Inv
- PCR Clean-up

### Sep 20, 2022:

- Colony PCR G3-1 and 7 G6 + gel
- [FACS] Electroshocking bacteria on 96 Well plate

### Sep 21, 2022:

- Miniprep of STK parts for Echo and Gibson Paris\_Inv for seq
- [FACS] Electroshocking bacteria on 96 Well plate

### Sep 22, 2022:

- Echo prep STK

### Sep 27, 2022:

- Chronoamperometry Paris\_Inv - 1 hour

### Sep 28, 2022:

- STK 110 plating
- Chronoamperometry Paris\_Inv - 8 min

### Sep 29, 2022:

- Chronoamperometry - 6 hours and 12 hours
- Pduet Plate reader experiment

### Sep 30, 2022:

- Chronoamperometry pOxyS - 6 hours
- Chart characterization

### Oct 2, 2022:

- Plate Reader Output Experiment
- Activation Inverter PsoxR

### Oct 6, 2022:

- Chronoamperometry - 16 hours

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

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| Engineering |

### Modularity and Standardisation in genetic engineering

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| **The modularity and standardization of electronic components are central to the architecture of emerging electronic and digital technologies**. Following engineering modularity and standardization principles, we have conceived and created a **bidirectional bio-electrical communication platform** by both genetic and electrical engineering.  In our bio-electronic platform, not only do we apply engineering principles to biological systems, but, we go a step further by engineering bacterial populations to act as electronic components. We have designed and tested a library of parts and constructs that, when transformed into bacterial populations, behave analogously to electronic components. Building on the analogy from the world of electronics, **we see our collection like the electronic components on the shelves of a hardware workshop where electronic designers can swap resistors and capacitors in a circuit**.  Our main Human Practices takeaway, from [our interviews with Brian Ringley, Cesar Harada, and Xiao Xiao](https://2022.igem.wiki/paris-bettencourt/human-practices), was that to be useful and serviceable to the engineering community, our bio-electronic platform had to be **as modular as possible**. We have used engineering principles to achieve our engineering goal of modularity in our genetic constructs. |

### What

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| Our wetware platform is made of genetic constructs organized into three categories:  * The **INPUTs**: electrical to biochemical signal transduction. * The **PROCESSORs**: biochemical to biochemical signal transduction performing genetic logic   and/or   biosensor operations. * The **OUTPUTs**: biochemical to electrical signal transduction.  [Go to Parts Collection](https://2022.igem.wiki/paris-bettencourt/part-collection) |

The modularity of our composite parts is based on two systems:

1. The three Input, Processor, and Output components depend on a 3 plasmid system.
2. The connection between these components is established by a standardized signal transduction
   communication pipeline using a tunable combination of transcription factors, promoters, and their
   associated chemical inducers.

These properties are illustrated below:

[![Engeneering Diagram]({{url_for('static', filename='Engeneering_Diagram.png')}})](%7B%7Burl_for%28%27static%27%2C%20filename%3D%27Engeneering_Diagram.png%27%29%7D%7D)

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| 1. Multiple Construct Combination - pDUET plasmids |

The constructs in our collection can function independently or jointly, similar to electronic components that
can be used in isolation or combined into electronic circuits.

We iterated through two cycles of the Design-Build-Test-Learn (DBTL) cycle
which allowed us to demonstrate that our genetic constructs function independently using the V35 backbone
vector
- and jointly - using the pDUET backbone vectors.

### Iteration 1 of the DTBL cycle:

In this iteration we demonstrate that our construct dpB.002 (Bba\_K4216027) assembled in the Moclo-compatible V35
vector works as expected.

**Design:**

We designed constructs with the pyocyanin inducible promoter pSoxS (Bba\_K4216006) upstream of a
fluorescence report in the MoClo compatible V35 vector plasmid.

**Build:**

We transformed this plasmid in a *E. coli* DH5alpha strain.

**Test:**

We measured the dose response of the pSoxS promoter to the inducer pyocyanin.

**Reflection:**

The V35 backbone vector has the advantage of being a high-copy number plasmid, meaning the
construct is highly expressed. The disadvantage is that adding other types of plasmids to an *E. coli*
already
containing the V35 would be too burdensome for the cell. However, we wanted to combine different types of
plasmid within a cell to increase the modularity of our system. We enquired our instructors, who informed us
of
the existence of the pDuet vector plasmids designed the for coexpression of multiple plasmids at a time in
one
strain. We iterated through a second DTBL cycle to achieve our desired modularity using these vectors.

![Responsive image](https://static.igem.wiki/teams/4216/wiki/wiki/engineering/iter1.png)

![Responsive image](https://static.igem.wiki/teams/4216/wiki/wiki/engineering/iter2.png)

### Iteration 2 of the DTBL cycle:

**Design:**

In our design, we selected three pDuet vector plasmids - pCOLADuet, pCDFDuet and pACYDuet - and inserted three
constructs - dpB.003 (Bba\_K4216028), dpB.011 (Bba\_K4216036), and dpB.013 (Bba\_K4216038) - in each of the vectors (see [pDuet combinaison optimizationation](https://2022.igem.wiki/paris-bettencourt/pduet_combinaison)
. The genetic constructs carried by the vectors
activate each other through a combination of transcription factors, promoters and inducers inspired by the
Marionette system (2).

**Build:**

We modified the pDuet vectors to make them MoClo compatible. To achieve this, we extracted, by PCR
amplification, both the pDuet vectors without the LacI expression cassette and the sfGFP expression cassette
from the V35 vector. Simultaneously, using designed primers with floating overhangs in our extractions, we
added restriction sites for BsaI with overhangs compatible with the CIDAR moclo to all pDuet vectors. We
assembled the now MoClo-compatible pDuet vectors with the extracted sfGFP transcriptional unit using Gibson
assembly. Then, we assembled theMoClo-compatiblee pDuets with our constructs using Golden Gate cloning.

**Test:**

We tested the expression of different combinations of the pDuet vectors.

**Learn:**

Modular gene expression is possible using the pDuet vectors (**figure 1**). The combination here is the
pACYDuet\_Bettencourt for the Input, the pCDFDuet\_Bettencourt for the Processor, and the pCOLADuet\_Bettencourt
for the Output. This figure shows the successful induction of three pDuet plasmids as a dose response of the
Input activation. This shows that the Output expression correlates positively with the level of induction in
the Input plasmid.

**Reflection:**

Using the DTBL cycles we achieved our engineering goal of modularity allowing the constructs of our toolkit to
be expressed together or independently. Based on the highest expressing combination, we chose to establish a
standard for the pDuets for our Input, Processor, and Output components.

**Modulation:**

We assembled our constructs into three interchangeable vector plasmids, that can be combined together or
used seperately.

**Standardisation:**

Each genetic component is associated with a pDuet vector

* pACYDuet for the INPUT
* pCDF for the PROCESSOR
* pCOLA for the OUTPUT.

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| 2) Tunability of Expression |

In our three constructs three plasmid system, each component is tunable using chemical inducers. This means
that the expression of each construct can be fine-tuned to transmit the signal more or less strongly. Like
knobs on a modular synthesizer, multimodal modes of control of our circuits is possible. We designed the
signal transmission of the 3 plasmids via the Processor component based on the Marionette strains developed
by
Meyer et al. (2). This system consists of transcription factors, promoters and inducers with high range and
specificity. Out of the 12 sensor circuits established in this system we selected two for our system:

* The CinR+OCH14+ pCin for the communication between the Input and the Processor
* and the LuxRQ + OC6 + pLux for the communication between the Processor and the Output

We selected these systems because of their compatibility with the CIDAR MoClo kit (they can be found in the
CIDAR MoClo extension kit) (1).

Our engineering goal was to achieve modularity in the Processor component, the “computation” module of our
system. As the logic operator, this module can perform common genetic logic on the signal transmition, such
as
amplification, resistance, inhibition, and biosensing.

In our Signal Modulation experiment we show the use of this module which can act as a biosensor -activates
gene expression at the presence of a chemical- or as a signal amplificator/resistor -tunes the signal
strength.

[To the Signal Modulation
Experiment](https://2022.igem.wiki/paris-bettencourt/signal_modulation)

The Processor component can be interchanged with any synthetic biology logic operation of choice. This
allows
for a variety of biosensors to be built and added to the collection of Processor components.

In summary, the goal of modularity and standardisation in our system was achieve through the applications
of
engineering principles.

[To the Proof of Concept](https://2022.igem.wiki/paris-bettencourt/proof-of-concept)

**Modulation:**

We assembled our constructs into three interchangeable vector plasmids, that can be combined together or
used seperately.

**Standardisation:**

Each genetic component is associated with a pDuet vector

* pACYDuet for the INPUT
* pCDF for the PROCESSOR
* pCOLA for the OUTPUT.

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| References |

[1] \*\*CIDAR MoClo: Improved MoClo Assembly Standard and New *E. coli* Part Library Enable Rapid Combinatorial
Design for Synthetic and Traditional Biology. Iverson SV, Haddock TL, Beal J, Densmore DM. ACS Synth
Biol.\* 2015 Nov 4. doi: 10.1002/bit.25814. PubMed [PMID
26479688](https://www.ncbi.nlm.nih.gov/pubmed/26479688).

[2] Meyer, A.J. et al. (2019) ‘Escherichia coli “Marionette” strains with 12 highly optimized
small-molecule
sensors’, Nature Chemical Biology, 15(2), pp. 196–204. Available
at: [https://doi.org/10.1038/s41589-018-0168-3.](https://doi.org/10.1038/s41589-018-0168-3)

[3] “Modular.” \*Merriam-Webster.com Dictionary\*, Merriam-Webster,
https://www.merriam-webster.com/dictionary/modular. Accessed 11 Oct. 2022.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

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| Experiments and Results |

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| With the overarrching goal of prooducing a modular and scalable electrogenetic toolkit, we developped genetic construcs and custom hardware and software.  Experiments fall in the following categories: Genetic engieering where we designed and characterised genetic construcst.   A serie of experiments aiming at screening a library of *E. coli* promoter for Electro-Genetic sensitivity.   A serie of experiemtn to characterise our hardware and software |

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| **Experiments fall in the following categories:**  * Genetic engineering where we designed and characterised genetic construcst. * A serie of experiments aiming at screening a library of *E. coli* promoter for   Electro-Genetic   sensitivity. * A serie of experiemtn to characterise our hardware and software |

### Main Acheivements

1. We delivered  **[a
   characterised part collection](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27part-collection%27%29%20%7D%7D)** enabling Electro-genetics in iGEM.
2. We successfully **[screened a
   library of promoter in *E. coli* for Electrically inducible Gene expression](%7B%7B%20url_for%28%27electrical_screening%27%29%7D%7D)** and
   added 19 responsive promoters to our collection.
3. We developed specific and new **Hardware enabling
   Electro-genetics**, at low cost, in iGEM and in context
   beyond synthetic biology.
4. We **described and characterised genetic circuits performing
   electrical signal processing function** in
   *E. coli*.

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| Genetic engineering |

Here we seek to develop and characterise the 3-plasmid system part by part and as a system

### Input characterisation

The input is designed to receive a signal that is then transmitted to the processor. First, we
constructed the input to receive an electrical signal, and, in turn, activate the transcriptional unit coding
for the processor transcription factor (CinR); however, upon reviewing the literature, we later discovered a
different electrical inducible promotor known as pSoxS, a piocynin inducible promotor that have already been
used in iGEM, which we deciede to implement instead.

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| * [pSoxS - dpB.002](%7B%7B%20url_for%28%27pSoxS%27%29%7D%7D) * [pOxyS - dpB.004](%7B%7B%20url_for%28%27pOxyS%27%29%7D%7D) * [pNasA - dpB.006](%7B%7B%20url_for%28%27pNasA%27%29%7D%7D) |

### Processor characterisation

The processor is the part in our device responsible for transmitting the signal from the input
to the output. Any signal passing through the processor can be modulated, allowing for the same input to have
multiple different outputs. Our processor is comprised two main parts: a (1) promotor (Pcin), which responds
to the signal of the input and a (2) chemical inducer (OHC14) with a transcription unit created to communicate
the signal to the output via activation of its promotor (pLUX) with a transcriptional factor(LuxRQ).

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| * [A Simple Resistance - dpB.010   -Marionette strain](%7B%7B%20url_for%28%27processor_alone%27%29%7D%7D) |

### Output characterisation

The output is designed to transform the message from the processor into a signal readable to our
measurement tools. One output construct we designed is comprised of a promotor (pLuxR) sensitive to the
processor’s message, and a transcriptional unit expressing an RFP or a Lysis gene. (The lysis gene destroys
the cells, releasing a sufficient concentration of ions to read a signal on our potentiostat.)

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| * [Electrical   Resistance as   a measure of Growth](%7B%7B%20url_for%28%27Electrical_resistance_growth_mesure%27%29%7D%7D)* [pLux76B - dpB.013](%7B%7B%20url_for%28%27pLux86B%27%29%7D%7D)   * [Lysis System - dpB.014](%7B%7B%20url_for%28%27lysis_ouptut%27%29%7D%7D) |

### Multiple plasmid characterisation

The multi-plasmid device is made of 3 plasmids that each code for either the input, the
processor and the output. This characterisation was designed and purposed to prove that the 3 plasmids can
communicate with each other and that the signal they pass to each other can be modulated with chemicals
products.

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| * [pDuets Combinaison   optimisation](%7B%7B%20url_for%28%27pduet_combinaison%27%29%7D%7D) * [Signal Modulation](%7B%7B%20url_for%28%27signal_modulation%27%29%7D%7D) |

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| Electro-Resonsive Promtoters screening |

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| Hardware |

We screened 500 promoters in E. coli for there response to exposure to electrical signals.

We found 19 responsive promoters (8 induced and 11 repressed).

[To the part collection](https://2022.igem.wiki/paris-bettencourt/part-collection)

To interface population of engineered bacterial with electronic systems as well as to prototype
and run experiement, we delivered a working [Hardware Collection](https://2022.igem.wiki/paris-bettencourt/hardware_custom)

### Electro Library Screening

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| * [Screening of electrically active promoters](%7B%7B%20url_for%28%27electrical_screening%27%29%7D%7D) * [Simple Growth curve on M9](%7B%7B%20url_for%28%27vmem_od_on_m9%27%29%7D%7D) * [Impact on Growth of   electroshock](%7B%7B%20url_for%28%27growth_electroshock%27%29%7D%7D) * [Effect of electroshock on Vmem](%7B%7B%20url_for%28%27impact_on_Vmem%27%29%7D%7D) * [Pooled experiments on Flow   Cytometry](%7B%7B%20url_for%28%27template_experiment%27%29%7D%7D) |

### Hardware Experiments

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| * [High Throughput Electro Actuator (HTEA)](https://2022.igem.wiki/paris-bettencourt/HTEA)* [AC Dispatcher (ACD)](https://2022.igem.wiki/paris-bettencourt/ACDispatcher)   * [Electro-Micro-Slide](https://2022.igem.wiki/paris-bettencourt/Electro_Micro_Slide)   * [Conductivity and OD     Sensor](%7B%7B%20url_for%28%27EC_OD%27%29%7D%7D)   * [The Electro Planner (Control Sofware)](https://2022.igem.wiki/paris-bettencourt/software)   * [Dummy Cell](https://2022.igem.wiki/paris-bettencourt/hardware_custom)   * [IO Rodeo Potentiostat](https://2022.igem.wiki/paris-bettencourt/IO_Rodeo_Potentiostat) |

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## 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.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}