iGEM 2020 — Team Thessaly

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# Team:Thessaly/Notebook

Team: Thessaly - 2020.igem.org

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* [HOME(current)](https://2020.igem.org/Team%3AThessaly)
* ABOUT

  [OUR TEAM](https://2020.igem.org/Team%3AThessaly/Team)[ATTRIBUTIONS](https://2020.igem.org/Team%3AThessaly/Attributions)
* PROJECT

  [PROJECT DESIGN](https://2020.igem.org/Team%3AThessaly/Design)[PROJECT DESCRIPTION](https://2020.igem.org/Team%3AThessaly/Description)[EXPERIMENTS](https://2020.igem.org/Team%3AThessaly/Experiments)[PROOF OF CONCEPT](https://2020.igem.org/Team%3AThessaly/Proof_Of_Concept)[ENGINEERING](https://2020.igem.org/Team%3AThessaly/Engineering)[MODEL](https://2020.igem.org/Team%3AThessaly/Model)[NOTEBOOK](https://2020.igem.org/Team%3AThessaly/Notebook)[RESULTS](https://2020.igem.org/Team%3AThessaly/Results)
* PARTS

  [PARTS OVERVIEW](https://2020.igem.org/Team%3AThessaly/Parts)[PARTS COLLECTION](https://2020.igem.org/Team%3AThessaly/Part_Collection)[CHARACTERIZATION](https://2020.igem.org/Team%3AThessaly/Contribution)[MEASUREMENTS](https://2020.igem.org/Team%3AThessaly/Measurement)
* HUMAN PRACTICES

  [EDUCATION &
   PUBLIC ENGAGEMENT](https://2020.igem.org/Team%3AThessaly/Education)[INTEGRATED
  HUMAN PRACTICES](https://2020.igem.org/Team%3AThessaly/Human_Practices)[MENTAL HEALTH](https://2020.igem.org/Team%3AThessaly/Mental_Health)[INCLUSIVITY](https://2020.igem.org/Team%3AThessaly/Inclusion)[COLLABORATIONS](https://2020.igem.org/Team%3AThessaly/Collaborations)
* IMPLEMENTATION

  [IMPLEMENTATION OVERVIEW](https://2020.igem.org/Team%3AThessaly/Implementation)[SAFETY](https://2020.igem.org/Team%3AThessaly/Safety)
* [MEDAL CRITERIA(current)](https://2020.igem.org/Team%3AThessaly/medal-crit)
* [GIANT JAMBOREE(current)](https://2020.igem.org/Team%3AThessaly/jamboree/)

# Notebook

![](https://static.igem.org/mediawiki/2020/7/73/T--Thessaly--team-1.jpg)

December

* We finished the recruitment of our team!

![](https://static.igem.org/mediawiki/2020/d/da/T--Thessaly--lamb.jpg)

January

* Brainstorming!
* Searching for inspiration and a cause.

![](https://static.igem.org/mediawiki/2020/5/50/T--Thessaly--graph.jpg)

February

* More brainstorming!
* We prepared games about Synthetic Biology for the Athens Science Festival that unfortunately wasn't organized, due to the SARS-CoV-2 pandemic.
* Created a board game called “Syn Phase” for “Open Days” an event that the Department of Biochemistry and Biotechnology of the University of Thessaly organizes every March, inviting schools to experience the world of science. Unfortunately, in our country at that time lockdown began to withstand the spread of SARS-CoV-2, so the event did not take place.

March

* More brainstorming!
* We prepared games about Synthetic Biology for the Athens Science Festival that unfortunately wasn't organized, due to the SARS-CoV-2 pandemic.
* Created a board game called “Syn Phase” for “Open Days” an event that the Department of Biochemistry and Biotechnology of the University of Thessaly organizes every March, inviting schools to experience the world of science. Unfortunately, in our country at that time lockdown began to withstand the spread of SARS-CoV-2, so the event did not take place.

##### Finally, we chose our project!

* We talked with people from our environment, who suffer from IBDs, because we wanted to understand their problems.
* On March 26, 2020 our team attended the virtual webinar “Tyler IBD Education Day” organized by Crohn’s and Colitis Foundation.
* We made a list about who can give us more information for our project and where we should start.
* Discussed our project with Mr. Leonidas Anthopoulos, Associate Professor, Mayor's Special Advisor, Municipality of Trikala.

![](https://static.igem.org/mediawiki/2020/2/2b/T--Thessaly--20-21.jpg)

April

##### We made the decision to participate in the iGEM Competition two phase program

* We sent emails and made phone calls to Biotechnologists, Clinical Nutritionists and Gastroenterologists for help.
* Emailed Mr. Ashram Bin Islam from the University of Asia Pacific.
* Skyped with Dr. Ioannis Serafetinidis Director Gastroenterologist - ELPIS Clinic – Volos, Scientific collaborator of Athens Hospital and head of polyclinics MEDICAL, YASIS – Athens.
* Online game: Syn Phase
* Press Coverage Trikalino papaki 28/4/20

![](https://static.igem.org/mediawiki/2020/9/94/T--Thessaly--igem-log.png)

May

##### We designed our team logo!

* Mr. George Skretas has been the Principal Investigator of the Laboratory of Enzyme and Synthetic Biotechnology at the Institute of Biology, Medicinal Chemistry and Biotechnology of the National Hellenic Research Foundation, Greece, where he currently holds the rank of Research Assistant Professor helped us in several parts of our project.
* We sent emails to various specialists, in order to gain input regarding our project and made more phone calls to Biotechnologists, Clinical Nutritionists and Gastroenterologists to ask for help.
* We met Dr. Oikonomou Konstantinos MD, PhD, a gastroenterologist-hepatologist in Larissa.

* After contacting us, a High School in Thessaloniki - Chortiatis General High School wanted to publish an article about Synthetic Biology, the iGEM competition, and projects of other High School teams that participated in the competition in previous years, in their online school newspaper.

##### We finished the development of our experimental design

* We contacted the team of Bielefeld 2014 via email.

##### We organised a Theatrical play

* Press coverage:

  Larissanet 06/05/20
  Larisa New 6/5/20
  Magnesia News 06/05/20
  ΝEWS1 12/05/20
  Taxydromos 13/05/20
  Limnos FM100 26/05/20

June

##### We designed our project logo!

![](https://static.igem.org/mediawiki/2020/0/01/T--Thessaly--amal.png)

##### We handed in the Safety Form for the Competition

* Created an interactive tutorial video for Synthetic Biology for the 1st elementary school Nea Maditos in Thessaloniki.

##### We started developing our experimental design

* We sent emails to various specialists, in order to gain input regarding our project.
* We made more phone calls to Biotechnologists, Clinical Nutritionists and Gastroenterologists to ask for help.
* We skyped with Ms. Adamantia Kyriakou, Associate Professor in Microbiology at Department of Nutrition and Dietetics at Harokopio University of Athens, who helped us to the biotechnological part of our project and with Ms. Kontogianni, Assistant Professor at Harokopio University of Athens, Department of Nutrition and Dietetics, Clinical Nutrition, who informed us regarding her scientific field.
* Sent emails to Greek organizations about IBDs.
* We organised the program for our Big Greek Meetup.

##### We finished the designing of our constructs and ordered the materials that we needed for our experiments

July

#####

* We sent emails to various specialists, in order to gain input regarding our project and made more phone calls to Biotechnologists, Clinical Nutritionists and Gastroenterologists to ask for help.
* Members of our team met a Member of the Board of Directors and representative of the Region of Thessaly, Mr. Costas Demertzis and discussed with him their problems, the treatment they receive, and our project.
* We met Dr. Paroutoglou George, Director of the Infusion Unit of the Gastroenterology Clinic of the General Hospital of Larissa George Paroutoglou and Dr. Helen Papadimitriou a specialist for IBDs from the General Hospital of Larissa
* We finalised the program for our Big Greek Meetup.

##### We hosted our Big Greek Meetup at 24-25-26/6/20

![](https://static.igem.org/mediawiki/2020/6/6c/T--Thessaly--meet-up.jpg)* We contacted the iGEM team from Ohio State University, in order to help us design our kill-switch module.
* We discussed our ideas with the Dietician, Nutritionist in Larissa Mr. Mekras Apostolis.
* We trained for our lab protocols.

August

* We sent emails to various specialists, in order to gain input regarding our project and made more phone calls to Biotechnologists, Clinical Nutritionists and Gastroenterologists to ask for help.

##### A week-long tribute 9-17/8/20 about Women in Science

* Our team communicated with the European Federation of Crohn's and Colitis Association (EFCCA).
* We designed a poster with a QR code to boost morale, strengthen the self-esteem and the self-confidence of people who suffer from IBDs.
* We created questionnaires that were distributed to the patients from the General Hospital of Larissa, with the help of Dr. Helen Papadimitriou.

##### We began our experiments in the lab

September

#####

* We got the questionnaires that were distributed to the patients from the General Hospital of Larissa, with the help of Dr. Helen Papadimitriou.
* We handed in the Safety Form for the Competition.

October

##### We performed our experiments for Characterization and tested the Prom Module, conducting, at the same time, our Proof of Concept

* Press coverage Kriti TV (Kalo Mesimeri) 15/10/20

##### We created a video that’s available on our online platforms, to inform the public about nutrition and health

* We organised a Presentation about Synthetic Biology and IBDs for schools.

##### We created our Presentation video

To Be Continued...

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#### Contact

* igemthessaly@gmail.com

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# Team:Thessaly/Engineering

Team: Thessaly - 2020.igem.org

[![alternative](https://static.igem.org/mediawiki/2020/d/d5/T--Thessaly--Transparent-callig.jpg)](https://2020.igem.org/Team%3AThessaly)

* [HOME(current)](https://2020.igem.org/Team%3AThessaly)
* ABOUT

  [OUR TEAM](https://2020.igem.org/Team%3AThessaly/Team)[ATTRIBUTIONS](https://2020.igem.org/Team%3AThessaly/Attributions)
* PROJECT

  [PROJECT DESIGN](https://2020.igem.org/Team%3AThessaly/Design)[PROJECT DESCRIPTION](https://2020.igem.org/Team%3AThessaly/Description)[EXPERIMENTS](https://2020.igem.org/Team%3AThessaly/Experiments)[PROOF OF CONCEPT](https://2020.igem.org/Team%3AThessaly/Proof_Of_Concept)[ENGINEERING](https://2020.igem.org/Team%3AThessaly/Engineering)[MODEL](https://2020.igem.org/Team%3AThessaly/Model)[NOTEBOOK](https://2020.igem.org/Team%3AThessaly/Notebook)[RESULTS](https://2020.igem.org/Team%3AThessaly/Results)
* PARTS

  [PARTS OVERVIEW](https://2020.igem.org/Team%3AThessaly/Parts)[PARTS COLLECTION](https://2020.igem.org/Team%3AThessaly/Part_Collection)[CHARACTERIZATION](https://2020.igem.org/Team%3AThessaly/Contribution)[MEASUREMENTS](https://2020.igem.org/Team%3AThessaly/Measurement)
* HUMAN PRACTICES

  [EDUCATION &
   PUBLIC ENGAGEMENT](https://2020.igem.org/Team%3AThessaly/Education)[INTEGRATED
  HUMAN PRACTICES](https://2020.igem.org/Team%3AThessaly/Human_Practices)[MENTAL HEALTH](https://2020.igem.org/Team%3AThessaly/Mental_Health)[INCLUSIVITY](https://2020.igem.org/Team%3AThessaly/Inclusion)[COLLABORATIONS](https://2020.igem.org/Team%3AThessaly/Collaborations)
* IMPLEMENTATION

  [IMPLEMENTATION OVERVIEW](https://2020.igem.org/Team%3AThessaly/Implementation)[SAFETY](https://2020.igem.org/Team%3AThessaly/Safety)
* [MEDAL CRITERIA(current)](https://2020.igem.org/Team%3AThessaly/medal-crit)
* [GIANT JAMBOREE(current)](https://2020.igem.org/Team%3AThessaly/jamboree/)

# Engineering

#### Overview

To tackle malnutrition and IBDs, we need a robust and comprehensive design to account both for project and experimental challenges. Thus, we have used the engineering principles of Abstraction Hierarchy and Modularity, and have split our project into two systems, the Detection and Therapeutic System. Within each system, there are several modules and devices that help us demonstrate the functionality of the project in an iterative and realistic manner. Explore our engineering design below.

#### -Modular Cloning/Expression Vectors:

Amalthea is composed of many systems, modules and devices, which translates to a lot of transcriptional units (TUs) that need to be cloned in appropriate vectors in the lab. We created six cloning/expression vectors, combining a bacterial-expression variation of the GoldenBraid standard (Sarrion-Perdigones et al. 2013), with the help of SEVA plasmids.

Click here to read more.

We designed a GoldenBraid-based vector toolkit aiming to adjust the GoldenBraid DNA assembly method for heterologous expression of proteins in bacteria, since it was originally developed for plants. For this purpose, we created proper customized
α (alpha)& Ω (omega) GB vectors, using the SEVA plasmid collection, to allow modular cloning and expression in bacteria.

**Go to results:** [Click here!](https://2020.igem.org/Team%3AThessaly/Results)

pDGB1 alpha 1R (α1R): [BBa\_K3505008](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505008)

pDGB1 alpha 2 (α2): [BBa\_K3505009](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505009)

pDGB1 omega 1R (Ω1R): [BBa\_K3505010](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505010)

pDGB1 omega 2 (Ω2): [BBa\_K3505011](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505011)

The SEVA collection is a plasmid repository for complex prokaryotic phenotypes. The collection entails most antibiotic resistance genes and ORIs, which can be used in any combination using rate restriction enzyme sites.This way, deconstruction and reconstruction of a plasmid and repurposing (e.g. transfer of a 5 TU construct from a cloning vector to an expression vector) is simple. (Martínez-García et al. 2020).

We used SEVA43 and SEVA23 as backbones to create the alpha and omega vectors. The alpha vectors have Kanamycin Resistance and the Omega vectors have Spectinomycin Resistance, to follow the GoldenBraid standard. pBBR, a low/medium copy ORI, was chosen to facilitate heterologous protein expression. However, due to low yield of the plasmid, downstream cloning was hindered and difficulty was increased. In our next Design-Build-Test cycle, We will use the modularity of the SEVA backbones to replace the low copy ORI by a high copy (pRO1600/ColE1).

Using our system, the whole SEVA collection can be utilized to create vectors with a desirable of antibiotic resistance genes and ORIs by targeting the T0 and T1 terminators that are universal.

**Go to Parts overview:** [Click here!](https://2020.igem.org/Team%3AThessaly/Parts)

#### “Detection System”

**Overview:** The purpose of the Detection System is to provide real-time monitoring of intestinal health, using Short-Chain Fatty Acids (SCFAs) as a biomarker.

#### -GPCR-Tango Module:

To detect clinically relevant concentrations of SCFAs, we designed a synthetic GPCR coupled with a NOT-GATE device. By combining a bacterial version of the Tango Assay (Dogra, Sona, Kumar and Yadav, 2016) and an inversion of signal output, we managed to detect the absence of SCFAs.

Click here to read more.

To achieve clinically relevant detection of SCFAs (butyrate, propionate, acetate) absence with one component, we designed a GPCR-mediated NOT-GATE module, which combines a typical NOT-GATE device with a bacterial version of the Tango Assay , which is mainly used in eukaryotes. The FFAR2 GPCR (*Kaemmerer*, 2010) is shown to bind SCFAs and facilitate downstream signalling *in vivo*

This module consists of four transcription units:

**1.** an inducible promoter for arabinose (ParaBAD):GPCR FFAR2: a V2 tail: a cleavage site-TCS:LacI: double terminator

[BBa\_K3505025](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505025)

**2.** an inducible promoter for arabinose (ParaBAD):b-arrestin:TEV protease, terminator

[BBa\_K3505026](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505026)

**3.** an constitutive promoter (AndersonJ23115):lac operator and reporter gene - (CFP):double terminator

[BBa\_K3505032](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505032)

**4.** a constitutive promoter (AndersonJ23115) :RraA :double terminator

[BBa\_K3505038](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505038)

The use of an inducible promoter provides a most reversible and flexible gene circuit and at the same time exhibits a higher efficiency and lower side effects as cell death. (Kallunki, Barisic, Jäättelä and Liu, 2019).

![alternative](https://static.igem.org/mediawiki/2020/e/e2/T--Thessaly--engin-img-01.png)

Figure 1 : GPCR -Tango Module

The existence of V2 tail permits the recruitment of a b-arrestin , that is combined with a TEV protease that recognizes and cleaves the cleavage site. When TEV protease cleaves , the lac repressor is released and binds to the lac operator ,inhibiting the transcription of reporter gene. In the presence of SCFAs the GPCR is activated.
Membrane proteins(MP) are pharmaceuticals targets and have a crucial role in cellular functions. However , MP overexpression result in cytotoxicity and cell death. In order to prevent that , we decide to co-express rraA , resulting in a dual positive effect on recombinant GPCR expression :

**1.** the expression of rraA , suppresses the cytotoxicity , thus resulting in enhanced levels of expression and bacterial biomass
**2.** contribute to cellular accumulation and result in better-folding of a MP (prokaryotic and eukaryotic origin)

*(Gialama et al., 2017)*

This module , due to an error in the orientation of omega1R , isn’t complete.
This construct isn’t the final one. The final construct was placed on an alpha1 vector which would consist of 2 omegas: omega1 and omega2. The construction of omega 1, was wrong, so we created omega1R. We didn’t have enough time to repeat vector construction experiments this year. Our purpose is to assemble and test the functionality of the GPCR-Tango Module, once we have access to the lab and report our results in iGEM 2021. See you in Paris!

#### -Tet-off module

To reduce the complexity and liabilities of bacterial GPCR expression, we designed a simple anhydrotetracycline (aTC)-induced NOT-GATE device, that has the same logical structure as the GPCR-Tango module, but uses the well-characterized transcriptional factor Tetracycline Repressor (TetR) (Gossen and Bujard, 1993).

Click here to read more.

This iGEM year was unique in many aspects. Because of that, we didn’t test all the devices/modules that we designed and built. However, we managed to do some initial characterization on our lower complexity devices. We designed and built a simple TetR-based on the NOT-GATE to prove the functionality of the device in our hands, before moving on to a SCFA-induced module.

In the presence of the strong chemical repressor anhydrotetracycline (aTC), TetR inhibition is blocked and the transcription of LacI is allowed. LacI in turn binds to Lac Operator (LacO) operator and inhibits the expression of a reporter gene.

In absence of aTC, TetR binds to the tetracycline response element-TRE and inhibits LacI expression. This inhibition allows the expression of the reporter gene. To obtain quantifiable and visual output, we used eCFP as a reporter gene.

**1.** constitutive promoter (AndersonJ23115) : Tet repressor : double terminator
[BBa\_K3505033](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505033)

**2.** constitutive promoter (AndersonJ23115) : Tet operator : lac repressor : double terminator
[BBa\_K3505034](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505034)

**3.** constitutive promoter (AndersonJ23115) :lac operator : ECFP: double terminator
[BBa\_K3505032](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505032)

**4.** stuffer

![alternative](https://static.igem.org/mediawiki/2020/c/c2/T--Thessaly--engin-img-02.png)

Figure 2 : Tet-off module

Our initial design included eGFP as the reporter gene. When we completed the cloning experiments, we immediately started plate-reader assays before sequencing results arrived. No detectable fluorescence was produced in our initial experiments testing the construct LacO-eGFP.

After sequencing results came in, we noticed some consistent single-point mutations in our positive eGFP clones, which when BLASTed returned eCFP as the best alignment. It turns out that, due to wrong starting template, we had cloned a eCFP instead of eGFP, which was the reason for the non-detectable fluorescence.
After adapting the plate-reader parameters to fit eCFP excitation/emission spectra, fluorescence was produced from the bacteria, expressing the construct.

This module, due to an error in the orientation of omega1R , isn’t complete.
This construct isn’t the final one. The final construct was placed on an alpha1 vector which would consist of 2 omegas: omega1 and omega2. The construction of omega 1, was wrong, so we created omega1R. We didn’t have enough time to repeat vector construction experiments this year. Our purpose is to assemble and test the functionality of the Tet-off Module, once we have access to the lab and report our results in iGEM 2021.

**To our results:** [Click here](https://2020.igem.org/Team%3AThessaly/Results)

#### Prom Module

To provide a failsafe in case the GCPR-Tango Module is not functional, we designed a promoter-based module for SCFAs detection. We used SCFA-induced promoters, pFliC (Tobe, Nakanishi and Sugimoto, 2010) and prpBCDE (Tsang, Horswill and Escalante-Semerena, 1998) to allow SCFAs to act as an input to the NOT-GATE device.

Click here to read more.

The next step in our design, was a NOT-GATE device that is activated by SCFAs. To do that, we integrated a set of SCFA-inducible promoters to regulate the expression of LacI.
In the presence of the SCFAs, LacI is activated and binds to Lac Operator (LacO) operator, inhibiting the expression of a downstream reporter gene.

In absence of SCFAs, LacI inhibition is blocked and transcription of the reporter gene takes place. To obtain quantifiable and visual output, we used eCFP as a reporter gene to obtain comparable data with our Tet-Off Module.

**1.** inducible promoter (pFliC) :: lac repressor : double terminator
[BBa\_K3505027](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505027)

**2.** constitutive promoter (AndersonJ23115) :lac operator: ECFP: double terminator
[BBa\_K3505032](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505032)

![alternative](https://static.igem.org/mediawiki/2020/7/77/T--Thessaly--engin-img-04.png)

Figure 4 :Prom module. pFliC induced from Butyrate

![alternative](https://static.igem.org/mediawiki/2020/b/b5/T--Thessaly--image-05.png)

Figure 5 :Prom module prpBCDE induced from Acetate and Propionate.

**To our proof of concept:** [Click Here](https://2020.igem.org/Team%3AThessaly/Proof_Of_Concept)

#### -Tyrosine-Tyrosinase Reporter Module

To obtain an output that can be easily digitized, we integrated a Tyrosine-Tyrosinase Reporter Module to couple SCFAs detection with an electrochemical signal. This can be achieved by the SCFA-induced overexpression of key Tyrosine metabolism enzymes.

Click here to read more.

For our Reporter Module, we made use of the ability of L-Dopa to produce electrochemical signal using a recently reported Tyrosine-Tyrosinase module. The electrochemical signal was chosen because of its high quality, easier digitalization and more accurate transmission to an external user (*VanArsdale et al., 2020*).

Adhesin Involved in Diffuse Adherence (AIDA) consists of three parts, Signal Peptide, Passenger and the transmembrane region. (Hörnström, Larsson, van Maris and Gustavsson, 2019)

![alternative](https://static.igem.org/mediawiki/2020/6/62/T--Thessaly--img-06.png)

Figure 6 : Tyrosinase converting L-Tyrosine to L-Dopa and L-Dopa quinone.

The signal peptide is cleaved, in order for the protein to be translocated to the membrane, while the Tyrosinase, that we add, acts as a Passenger. We chose the Tyr1 gene coding Tyrosinase because it is the smallest of all Tyrosinases available and lacks cysteines, resulting into an easier transportation to the outer membrane. (*Gustavsson et al., 2016*)

We used a constitutive promoter for the regulation of the Tyr1-AIDA. Having in mind the concept that Tyrosinase must be there waiting the L-Tyrosine to appear. This led to reduced cell growth and reduced expression of the protein, which we hypothesized is caused by toxicity. In the next Design-Build-Test cycle, we will add an inducible promoter and express antitoxic proteins (*Gialama et al., 2017*) for better expression.

**To our results:** [Click here](https://2020.igem.org/Team%3AThessaly/Results)

#### “Therapeutic System”

The purpose of the therapeutic system is to provide the essential elements that IBDs patients lack and relieve their symptoms – SCFAs.

**SCFAs production:** We integrate metabolic pathways for three key SCFAs – butyrate, propionate, acetate – in *E. coli* to overproduce each one separately. The production from the bacteria will take place either as probiotics producing in *vivo* the SCFAs, or ex *vivo* and then oral consumption as a prebiotic.

Click here to read more.

Our goal is the production of SCFAs, in order to replenish the missing amount of them. To achieve that, we tap into common pathways of metabolism (e.g Glycolysis , Krebs Cycle) and engineered them.

For the production of acetate: We overexpress the genes of the C2-fermentive pathway that leads to the formation of acetate. When acetyl-CoA is formed we convert it to acetate by overexpressing 2 genes: Pta - Phosphate Acetyltransferase and AckA – Acetate Kinase. (Miscevic et al., 2018)

For the production of propionate : We tap into the Tricarboxylic Acid Cycle , and when the succinyl-CoA is formed , we convert it to propionate adding 3 genes from the Sleeping Beauty Operon. The Sleepign beauty mutase (Sbm) Operon , in *E.coli* , contains genes similar to those that take part in the oxidative propionate pathway of Propionobacteria. :*Sbm – methylmalonyl-CoA mutase, YgfG: methylmalonyl- CoA decarboxylase* and last *YgfH: propionyl-CoA/succinyl- CoA transferase* (Akawi et al., 2015).above-heading

For the production of butyrate : we tap into glycolysis and when acetyl-CoA is formed with the use of 6 genes we convert it to butyrate : BktB - β-ketothiolase , Hbd - (S)-3HB-CoA dehydrogenase , Crt - Crotonase , Ter - trans-2-enoyl-CoA re- ductase , Ptb- phosphate butyryltransferase , Buk - butyrate kinase. Some of the genes we add, are from Butyrogenic Bacteria, such as Clostridium acetobutylicum and Treponema denticola. (Miscevic et al., 2018)

![alternative](https://static.igem.org/mediawiki/2020/8/8e/T--Thessaly--engin-img-07.png)

Figure 7 : Production of SCFAs taking advantage of common metabolic Pathways.

Optimization of SCFAs production: To optimize SCFAs production, knockouts of key enzymes are needed to redirect the carbon flux appropriately. We will design knockout experiments using CRISPR/Cas9, ssDNA recombineering or a combination of the two to obtain strains optimized for SCFAs production. Stay tuned!

#### “Dry Lab”

For the evaluation of the patient’s microbiome, we decided on a non-invasive approach that will bring no pain or discomfort to the patient, while also presenting effective and safe transmission of the data to the experts. Thus, we agreed upon using an ingestible capsule, which is rendered safe and effective by applying the following materials in its manufacturing process.

Click here to read more.

The capsule and its bioelectronic system is separated in two channels. The first channel contains the detection bacteria with the semipermeable membrane. We selected an ABS plastic (Protolabs Inc.) Semipermeable membrane (0.22μm pore size, EIMF22205, Millipore Sigma) for the purposes of our project (Mimee et al., 2018).

The bacterial channel is connected with the electronic channel via the detection electrodes. We selected a CHI101 ⌀2mm gold working electrode, a CHI115 platinum counter electrode and a CHI 111 Ag/AgCl reference electrode from CH Instruments. Those specific electrodes were picked according to papers that support their effectiveness at tyrosine/tyrosinase reactions (VanArsdale et al., 2020).

A custom PCB (Advanced Circuits) was selected as a board for the electronics, while a basic PIC18F Microcontroller is employed for the calculations and control of the capsule’s function. The microcontroller works well with our capsule requirements, as it is light, has a low power consumption and works effectively in the temperature of the human body.

For the production of butyrate : we tap into glycolysis and when acetyl-CoA is formed with the use of 6 genes we convert it to butyrate : BktB - β-ketothiolase , Hbd - (S)-3HB-CoA dehydrogenase , Crt - Crotonase , Ter - trans-2-enoyl-CoA re- ductase , Ptb- phosphate butyryltransferase , Buk - butyrate kinase. Some of the genes we add, are from Butyrogenic Bacteria , such as Clostridium acetobutylicum and Treponema denticola. (Miscevic et al., 2018)

Two Duracell Silver Oxide 364 batteries with supply voltage of 1.5 V each, electric charge of ~80mAh and a maximum current of 70mA were picked as a power source for the capsule.

4-15μm of Parylene-C (Daisan Kasei Co.) was selected as coating for the electronics and also as a moisture barrier (Mimee et. al, 2018).

As for the antenna, it should be comprised of materials with high relative permittivity, medium conductivity, and preferably biocompatible. For a biocompatible encapsulation we have chosen Al2O3 (Degussit AL23, εr =9.9, tanδ = 0.0001) as practically lossless, biocompatible, and widely available commercially in various shapes and sizes (Nikolayev et al., 2017) .

For the substrate, we picked FR-4(εr = 4.8, tanδ = 0.026), as it is the most prevalent material in all the antenna designs that we studied and also very flexible to help roll the antenna easier within the capsule.

The ground plane and patch are made of annealed copper, due to its high electric conductivity and low cost.
The antenna modeling took place within the AntennaDesigner toolkit of MATLAB.

##### References

Dogra, S., Sona, C., Kumar, A. and Yadav, P., 2016. Tango assay for ligand-induced GPCR–β-arrestin2 interaction. Methods in Cell Biology, pp.233-254.

Gialama, D., Delivoria, D., Michou, M., Giannakopoulou, A. and Skretas, G., 2017. Functional Requirements for DjlA- and RraA-Mediated Enhancement of Recombinant Membrane Protein Production in the Engineered *Escherichia coli* Strains SuptoxD and SuptoxR. Journal of Molecular Biology, 429(12), pp.1800-1816.

Gossen, M. and Bujard, H., 1993. Anhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells. *Nucleic Acids Research*, 21(18), pp.4411-4412.

Gustavsson, M., Hörnström, D., Lundh, S., Belotserkovsky, J. and Larsson, G., 2016. Biocatalysis on the surface of *Escherichia coli*: melanin pigmentation of the cell exterior. *Scientific Reports*, 6(1).

Hörnström, D., Larsson, G., van Maris, A. and Gustavsson, M., 2019. Molecular optimization of autotransporter-based tyrosinase surface display.  *Biochimica et Biophysica Acta (BBA) - Biomembranes*, 1861(2), pp.486-494.

Kaemmerer, E., 2010. Fatty acid binding receptors in intestinal physiology and pathophysiology. *World Journal of Gastrointestinal Pathophysiology*, 1(5), p.147.

Kallunki, Barisic, Jäättelä and Liu, 2019. How to Choose the Right Inducible Gene Expression System for Mammalian Studies?. *Cells*, 8(8), p.796.

Mimee, M., Nadeau, P., Hayward, A., Carim, S., Flanagan, S., Jerger, L., Collins, J., McDonnell, S., Swartwout, R., Citorik, R., Bulović, V., Langer, R., Traverso, G., Chandrakasan, A. and Lu, T., 2018. An ingestible bacterial-electronic system to monitor gastrointestinal health. *Science*, 360(6391), pp.915-918.

Nikolayev, D., Zhadobov, M., Le Coq, L., Karban, P. and Sauleau, R., 2017. Robust Ultraminiature Capsule Antenna for Ingestible and Implantable Applications. IEEE *Transactions on Antennas and Propagation*, 65(11), pp.6107-6119.

Tobe, T., Nakanishi, N. and Sugimoto, N., 2010. Activation of Motility by Sensing Short-Chain Fatty Acids via Two Steps in a Flagellar Gene Regulatory Cascade in Enterohemorrhagic *Escherichia coli. Infection and Immunity*, 79(3), pp.1016-1024.

Tsang, A., Horswill, A. and Escalante-Semerena, J., 1998. Studies of Regulation of Expression of the Propionate (prpBCDE) Operon Provide Insights into How Salmonella typhimurium LT2 Integrates Its 1,2-Propanediol and Propionate Catabolic Pathways. *Journal of Bacteriology*, 180(24), pp.6511-6518.

VanArsdale, E., Hörnström, D., Sjöberg, G., Järbur, I., Pitzer, J., Payne, G., van Maris, A. and Bentley, W., 2020. A Coculture Based Tyrosine-Tyrosinase Electrochemical Gene Circuit for Connecting Cellular Communication with Electronic Networks. *ACS Synthetic Biology*, 9(5), pp.1117-1128.

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# Team:Thessaly/Experiments

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# Experiments

Our work in the lab was based on standard protocols and general experimental
procedures. The workflow of our experiments can be summarized in three main
steps:

* Cloning of the genetic material into the desirable plasmid
* Production of protein from target genes cloned in appropriate expression vectors
* Proof of concept

To read the in-depth protocols used in our project, see the categories below. Moreover , in this page you can find our LabBook with tips or adaptations we have made according to our needs.

General lab protocols

1) General lab protocols : common recipes
 we use while being in lab (media-buffer)

##### Media

Liquid and solid preparation specifically for the growth and storage of microorganisms and other type of cells.
A variety of media can be used for different experiments or strains.

##### Buffer

The main role of the buffer solution is to maintain the pH around the optimal level, suitable for the growth of bacterial cultures.

Ethinium bromide, 10mg/ml

* Dissolve 0.2 g ethidium bromide in 20 ml H2O.
* Mix thorougly
* Store at 4°C in dark
* CAUTION: Ethidium bromide is a mutagen and must be handled carefully.

TAE (Tris/acetate/EDTA) electrophoresis buffer

**50x stock solution:**

* 242 g Tris base
* 57.1 ml glacial acetic acid
* 37.2 g Na2EDTA⋅2H2O
* H2O to 1 liter

**Working solution, pH ~8.5:**

* 40mM Tris⋅acetate
* 2 mM Na2EDTA⋅2H2O
  (add 980ml DI Water to 20ml 50X TAE to make 1X TAE)

-To make 1x TAE from 50x TAE : add 980 ml dh2O and 20ml from 50x TAE

TBE (Tris/borate/EDTA) electrophoresis buffer

**10x stock solution, 1 liter:**

* 108 g Tris base (890 mM)
* 55 g boric acid (890 mM)
* 40 ml 0.5 M EDTA, pH 8.0

Antibiotic stock

**Kanamycin stock 50mg/mL**

* Dissolve 0,5 g of Kanamycin in 10mL ddH2O
* Mix thoroughly
* Filter into 2mL eppendorf tubes by using a 22.μm filter for sterilization
* Store at -20oC

**Chloramphenicol stock 35mg/mL**

* Dissolve 0.34 g of Chloramphenicol in 10mL Ethanol 100%
* Mix thoroughly
* Filter into 2mL eppendorf tubes by using a 22.μm filter for sterilization
* Store at -20oC

**Streptomycin/Spectomycin**

* Dissolve 1g of Streptomycin/ Spectomycin in 10mL ddH2O
* Mix thoroughly
* Filter into 2mL eppendorf tubes by using a 0.22μm filter for sterilization
* Store at -20oC

Inoue transformation buffer

|  |  |  |
| --- | --- | --- |
| Reagent | Amount/L | Final concentration |
| MnCl2.4 H2O | 10.88 g | 55 mM |
| CaCl2.2H2O | 2.20 g | 15 mM |
| KCI | 18.65 g | 250 mM |
| PIPIES (0.5 M, pH 6.7) | 20 ml | 10 mM |
| H2O | to 1 L |  |

→ -Do not sterilize in a autoclave! Use a 0.44 μm filter for sterilization.

M9 Medium

**Preparing the 5x stock:**

**Add the following reagents to a flask:**

* 64 g Na2HPO4, seven hydrate
* 15 g KH2PO4
* 5 g NH4Cl
* 2.5 g NaCl
* 1 liter of high-quality distilled water

-Heat with stirring until the components are dissolved

-Sterilization : use a autoclave at 15 lb/in2 for 15 min

-Add antibiotics after the autoclave cycle and wait until the bottle is less than 50oC (capable to touch the bottom of the flask/Duran for 10 seconds )

**Preparing the 1x stock :**

**For 1x stock of M9 medium :**

To make 1X working solution, the 5X media should be diluted to 1× with high quality sterile distilled water

* 200ml 5x stock
* 1 ml 1 M MgSO4.7H2O
* 10 ml 20% D-glucose
  Typically, several additional components are also added to make a complete medium.
* 34 ml 0.5% vitamin B1 (thiamine)
* 10 ml 20% Casamino Acids
* 5 μl 1Μ CaCl2
* Antibiotic for selection

-Sterilization : use a 0.44μm filter

Tyrosinase assay Buffer

**Preparing the 50x stock :**

* 2,5M Tris-HCL
* 0,5M CuSO4 (pH 7.5)

**-Preparing 2x stock for L-tyrosine**

* 2g/L L-tyrosine

**-For 1x stock:**

* Measure the appropriate μL from each one :
* 50mM Tris-HCL
* 10μM CuSO4 (pH 7.5)
* 1g/L L-tyrosine

-Mix well using a strirrer until the the components are dissolved.

- Sterilization 50x stock using a 0.22μ filter

Agarose Gel Preparation

**For 60ml of 1% w/v agarose we need:**

* 60ml 1X TAE (diluted from 50X TAE stock)
* 0,6gr agarose
* 2,1μl EtBr (3.6ul for 100mL gel, 5.4ul for 150mL gel)

**Instructions**

* **1.** Measure the appropriate volume of 1X TAE (falcon tube for the measurement) and 0,6gr of dry agarose.
* **2.** Mix TAE and agarose in a flask.
* **3.** Microwave for 2-3 mins (until it boils)
* **4.** Microwave for 2-3 mins (until it boils)
* **5.** When in the right temperature (not too hot), add 2,16μl EtBr. Pipette up and down and mix the flask to spread the EtBr everywhere.
* **6.** Prepare the gel box, combs and calibrate the scaffolds
* **7.** Add the liquid gel into the gel box.
* **8.** After 10-15mins, untighten the scaffold and remove the gel box with the gel still on.

Nanodrop

* Nanodrop is high quality spectrophotometer.
* Select DNA measurement ‘Nucleic Acid’ in the NanoDrop menu
* Clear the surface of the Nanodrop with dH2O
* Calibrate the spectrophotometer and blank with Elution Buffer
* Place 1 ul per sample on the Nanodrop and measure the concentration.
* At the end , clear the surface with dH2 O.

Agar plates

* 40g of medium
* 1L of dH2O

- Mix 40g of medium into 1L of dH2O.

- Heat with stirring until the components are dissolved (1 minute).

- Sterilization : use a autocleave at 121oC for 15 minutes.

- Add antibiotics after the autoclave cycle and wait until the bottle is less than 50oC (capable to touch the bottom of the flask/Duran for 10 seconds).

- Mix well and dispense into plates (apprx.30mL each one).

LB Liquid Cultures

* 25g of medium
* 1L of H2O

- Mix 25g of medium into 1L of dH2O.

- Mix well until the components are dissolved.

- Sterilization : use a autocleave at 121oC for 15 minutes.

- Add antibiotics after the autoclave cycle and wait until the bottle is less than 50oC (capable to touch the bottom of the flask/Duran for 10 seconds).

- Mix well and dispense into Eppendorfs.

Go Back

Lab protocols

2) Lab protocols : a list of instructions to
 perform an experiment

Innoue Chemically Competent

##### A. Preparation of cells

**1.** Prepare Inoue transformation buffer (chilled to 0 °C before use).

**2.** Organic contaminants in the H2O used to prepare transformation
buffers can reduce the efficiency of transformation of competent
bacteria. H2O obtained directly from a well-serviced Milli-Q filtration
system usually gives good results. If problems should arise, treat the
deionized H2O with activated charcoal before use.

**a.** Prepare 0.5 M PIPES (pH 6.7). Adjust the pH of the solution to 6.7 with 5
M KOH, and then add pure H 2 O to bring the final volume to 100 ml.
Sterilize the solution by filtration through a disposable prerinsed
Nalgene filter. Divide into aliquots and store frozen at -20 °C

**b.** Prepare Inoue transformation buffer by dissolving all of the solutes
listed below in 800 ml of pure H2O and then add 20 ml of 0.5 M PIPES
(pH 6.7). Adjust the volume of the Inoue transformation buffer to 1 liter
with pure H2O.

**c.** Sterilize Inoue transformation buffer by filtration through a prerinsed
0.45-mm Nalgene filter. Divide into aliquots and store at -20 °C.

**3.** Pick a single bacterial colony (2-3 mm in diameter) from a plate that has
been incubated for 16-20 h at 37 °C. Transfer the colony into 25 ml of
LB broth or SOB medium in a 250 ml flask. Incubate the culture for 6-8
h at 37 °C with vigorous shaking (250-300 rpm).

**4.** At about 6 o'clock in the evening, use this starter culture to inoculate
three 1-L flasks, each containing 250 ml of SOB. The first flask receives
10 ml of starter culture, the second receives 4 ml, and the third receives
2 ml. Incubate all three flasks overnight at 18-22 °C with moderate
shaking.

**5.** The following morning, read the OD 600  of all three cultures. Continue to
monitor the OD every 45 min.

**6.** When the OD 600 of one of the cultures reaches 0.55, transfer the culture
vessel to an ice-water bath for 10 min. Discard the two other cultures.

**7.** The ambient temperature of most laboratories rises during the day and
falls during the night. The number of degrees and the timing of the
drop from peak to trough varies depending on the time of year, the
number of people working in the laboratory at night, and so on.
Because of this variability, it is difficult to predict the rate at which
cultures will grow on any given night. Using three different inocula
increases the chances that one of the cultures will be at the correct
density after an overnight incubation.

**8.** Harvest the cells by centrifugation at 2,500 x g (3,900 rpm in a Sorvall
GSA rotor) for 10 min at
4 °C.

**9.** Pour off the medium and store the open centrifuge bottle on a stack of
paper towels for 2 min. Use a vacuum aspirator to remove any drops of
remaining medium adhering to walls of the centrifuge bottle or
trapped in its neck.

**10.** Resuspend the cells gently in 80 ml of ice-cold Inoue transformation
buffer. The cells are best suspended by swirling rather than pipetting or
vortexing.

**11.** Harvest the cells by centrifugation at 2,500 x g (3,900 rpm in a Sorvall
GSA rotor) for 10 min at
 4 °C.

**12.** Pour off the medium and store the open centrifuge tube on a stack of
paper towels for 2 min.

**13.** Use a vacuum aspirator to remove any drops of remaining medium
adhering to the walls of the centrifuge tube or trapped in its neck.

##### B. Freezing of competent cells

**1.** Resuspend the cells gently in 20 ml of ice-cold Inoue transformation
buffer.

**2.** Add 1.5 ml of DMSO. Mix the bacterial suspension by swirling and then
store it in ice for 10 min.

**3.** Working quickly, dispense aliquots of the suspensions into chilled,
sterile microfuge tubes.

**4.**Immediately snap-freeze the competent cells by immersing the tightly
closed tubes in a bath of liquid nitrogen. Store the tubes at -70 °C until
needed. Freezing in liquid nitrogen enhances transformation efficiency
by ~5-fold. For most cloning purposes, 50 ml aliquots of the competent-
cell suspension will be more than adequate. However, when large
numbers of transformed colonies are required (e.g., when constructing
cDNA libraries), larger aliquots may be necessary.

**5.** When needed, remove a tube of competent cells from the -70 °C
freezer. Thaw the cells by holding the tube in the palm of the hand. Just
as the cells thaw, transfer the tube to an ice bath. Store the cells on ice
for 10 min.

**6.** Use a chilled, sterile pipette tip to transfer the competent cells to
chilled, sterile 17 x 100-mm polypropylene tubes. Store the cells on ice.
Glass tubes should not be used since they lower the efficiency of
transformation by ~10-fold.

##### C. Transformation

**1.** Include all of the appropriate positive and negative controls.

**2.** Add the transforming DNA (up to 25 ng per 50 ml of competent cells) in
a volume not exceeding 5% of that of the competent cells. Swirl the
tubes gently several times to mix their contents. Set up at least two
control tubes for each transformation experiment, including a tube of
competent bacteria that receives a known amount of a standard
preparation of superhelical plasmid DNA and a tube of cells that
receives no plasmid DNA at all. Store the tubes on ice for 30 min.

**3.** Transfer the tubes to a rack placed in a preheated 42 °C circulating
water bath. Store the tubes in the rack for exactly 90 sec. Do not shake
the tubes. Heat shock is a crucial step. It is very important that the cells
be raised to exactly the right temperature at the correct rate. The
incubation times and temperatures given here have been worked out
using Falcon 2059 tubes. Other types of tubes will not necessarily yield
equivalent results.

**4.** Rapidly transfer the tubes to an ice bath. Allow the cells to cool for 1-2
min.

**5.** Add 800 ml of SOC medium to each tube. Warm the cultures to 37 °C in
a water bath, and then transfer the tubes to a shaking incubator set at
37 °C. Incubate the cultures for 45 min to allow the bacteria to recover
and to express the antibiotic resistance marker encoded by the
plasmid. To maximize the efficiency of transformation, gently agitate
(<225 cycles/minute) the cells during the recovery period.

**6.** Transfer the appropriate volume (up to 200 ml per 90 mm plate) of
transformed competent cells onto agar SOB medium containing 20 mM
MgSO 4  and the appropriate antibiotic. When selecting for resistance to
tetracycline, the entire transformation mixture may be spread on a
single plate (or plated in top agar). In this case, collect the bacteria by
centrifuging for 20 sec at room temperature (RT) in a microfuge, and
then gently resuspend the cell pellet in 100 ml of SOC medium by
tapping the sides of the tube. IMPORTANT Sterilize a bent glass rod by
dipping it into ethanol and then in the flame of a Bunsen burner. When
the rod has cooled to RT, spread the transformed cells gently over the
surface of the agar plate. When selecting for resistance to ampicillin,
transformed cells should be plated at low density (<104colonies per 90
mm plate), and the plates should not be incubated for more than 20 h
at 37 °C. The enzyme b-lactamase is secreted into the medium from
ampicillin-resistant transformants and can rapidly inactivate the
antibiotic in regions surrounding the colonies. Thus, plating cells at high
density or incubating them for long periods of time results in the
appearance of ampicillin-sensitive satellite colonies. This problem is
ameliorated, but not completely eliminated, by using carbenicillin
rather than ampicillin in selective media and increasing the
concentration of antibiotic from 60 mg/ml to 100 mg/ml. The number
of ampicillin-resistant colonies does not increase in linear proportion to
the number of cells applied to the plate, perhaps because of growth-
inhibiting substances released from the cells killed by the antibiotic.

**7.** Store the plates at RT until the liquid has been absorbed.

**8.** Invert the plates and incubate them at 37 °C. Transformed colonies
should appear in 12-16 h.

Resuspension of gBlocks /primers

##### When still dry:

**1.** Spin down the pellet. You need everything to be on the tip.

**2.** Open the tube very carefully (don’t touch the lid from the inside).

**3.**Add appropriate volume of ddH2O (for primers :creating a concentration of
100pmol/μL).

**4.** Close the lid.

**5.** Vortex thoroughly (~20s).

**6.** Spin down.

**For primers:** To create the **working concentration 10pmol/μL or 10μM (1:10 of
stock):**

**7.** Dilute 10μL of stock concentration into 90μL ddH2O total volume is 100μL

PCR Kapa taq – Polymerase Chain Reaction Protocol with Kapa Taq DNA
Polymerase

##### 1. Set up a 10μL PCR reaction ( Keep all your reagents on ice ) :

● 1 μL template DNA (10Nng-500ng)
● 2.5μl 10X Taq buffer with MgCl2
● 0.5μl dNTP mix (10 mM each nt)
● 0.8μl Forward Primer (10μM stock)
● 0.8μl Reverse Primer (10μM stock)
● 0.1μl Taq DNA Polymerase (5 units/μl)
● 4.3μl Sterile dH2O (variable)

**2.** Place reaction tubes in PCR machine.

**3.** Set annealing temperature 5°C below the primer melting
temperature (Tm). Alternatively, use NEB’s Tm Calculator to
determine the optimal temperature.

**4.** Set extension step at 1 minute per kilobase of product depending on
whether you are using a polymerase with proofreading capabilities.

**5.** Initial Denaturation for 3 minutes at 95°C. 6. Denature for 30
seconds at 95°C

**6.** Denature for 30 seconds at 95°C.

**7.** Anneal primers for 30 seconds at 55°C (or 5°C below Tm).

**8.** Extend DNA for 2 minutes at 72°C.

**9.** Repeat steps 2-4 for 34 cycles.

**10.** Final Extension for 2 minutes at 72°C

**11.** Run on a gel to check a size and concertation of PCR product

##### **Note:** See manufacturer’s instructions for specific instructions about extension time and temperatures.

PCR protocol for NEB’s Q5 High-Fidelity 2X Master Mix

##### Reaction setup according to NEB

|  |  |  |
| --- | --- | --- |
| Component | 50 µl Reaction | Final Concentration |
| Q5 High-Fidelity 2X Master Mix | 25 µl | 1X |
| 10 µM Forward Primer | 2.5 µl | 0.5 µM |
| 10 µM Reverse Primer | 2.5 µl | 0.5 µM |
| Template DNA | 1 μl | 0,71 ng |
| Nuclease-Free Water | to 50 µl |  |

● Prepare all reactions on ice.
● Gently mix the reaction. Collect all liquid to the bottom of the tube by a
quick spin if necessary. Overlay the sample with mineral oil if using a PCR
machine without a heated lid.
● Transfer PCR tubes to a PCR machine and begin thermocycling.

##### Thermocycling Conditions for a Routine PCR

|  |  |  |
| --- | --- | --- |
| STEP | TEMP | TIME |
| Initial Denaturation | 98°C | 30 seconds |
| 25–35 Cycles | 98°C | 5–10 seconds |
|  | \*50–72°C | 10–30 seconds |
|  | 72°C | 20–30 seconds/kb |
| Final Extension | 72°C | 2 minutes |
| Hold | 4–10°C |  |

One-pot dig-lig reaction (Golden Gate/GoldenBraid)

We use the same protocol for different level assemblies, while changing the acceptor
vectors and the enzyme. Pay close attention to the enzyme you need to use each
time and always write it down in you labbook.

Total reaction volume: 10 μL

✔ 50-75 ng acceptor vector (pUPD2, p3alpha, p3omega etc.)
✔ 40-70 ng of each part (If you keep within this range and especially the upper
range, you will be fine most of the cases)
✔ 1 μL 10X T4 DNA ligase buffer
✔ 1 μL T4 DNA Ligase
✔ 0.5 μL restriction enzyme (BsmBI for L0, BsaI to alpha, BsmBI to omega)
✔ X μL ddH2O to reach 10 ul

You will need a thermocycler (PCR Machine) to run this protocol. The protocol we
use in our lab is termed “GBLong”:

**1.** 2 min at 37 degrees C (optimal temp for Restriction Enzymes).
**2.** 5 min at 16 degrees C (optimal temp for Ligase).
**3.** Repeat steps 1. and 2. 50 times.
**4.** 5 min at 80 degrees C (to kill enzymes).
**5.** Rest at 16 degrees C

Transformation Protocol

**1.** Take competent cells out of -80°C and thaw on ice (approximately 20 mins).
**2.** Mix 1μl of DNA (usually 10 pg - 100 ng) into 50-100 μL of competent cells in a
microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the
tube with your finger a few times.
**3.** Incubate the competent cell-DNA mixture on ice for 20-30 mins.
**4.** Heat shock each transformation tube by placing the bottom of the tube into
a 42°C water bath for 60 sec
**5.** Put the tubes back on ice for 2-5 min.
**6.** Add 900 μl LB (without antibiotic) to the bacteria and grow in 37°C shaking
incubator for 1h (under sterile conditions).
**7.** Centrifugation (9,000 x g for 1 min) and resuspend in a smaller volume of LB.
**8.** Plate some or all of the transformation onto a 10 cm LB agar plate containing
the appropriate antibiotic.
**9.** Incubate plates at 37°C overnight.

Restriction Digestion of DNA Protocol
Procedure

**1.** Select restriction enzymes to digest your plasmid.
**2.** Determine an appropriate reaction buffer by reading the instructions for your.
**3.** In a 1.5mL tube combine the following:

● DNA
● Restriction Enzyme(s)
● Buffer
● BSA (if recommended by manufacturer) [I never add BSA]
● dH2O up to total volume

Our restriction digestions are typically 10 to 20 uL in total volume. A typical reaction
is :

● 1 µg DNA
● 0.5 µL of each Restriction Enzyme
● 1 µL 10x Buffer
● x µL dH2O (to bring total volume to 30µL)

**4.** Mix gently by pipetting.
**5.** Incubate tube at appropriate temperature (usually 37 °C) for 1,5-2 hours.

Always follow the manufacturer’s instructions.
To visualize the results of your digest, conduct gel electrophoresis.

Gel extraction - DNA extraction from agarose gels

**1.Excise DNA fragment / solubilize gel slice**

**Note:** Minimize UV exposure time to avoid damaging the DNA. Refer to section 2.5
for more tips on agarose gel extraction.

-  Take a clean scalpel to excise the DNA fragment from an agarose gel.
-  Remove all excess agarose.
-  Determine the weight of the gel slice and transfer it to a clean tube.
-  For each 100 mg of agarose gel < 2 % add 200 μL Buffer NTI.
-  For gels containing > 2 % agarose, double the volume of Buffer NTI.
-  Incubate sample for 5–10 min at 50 °C.
-  Vortex the sample briefly every 2–3 min until the gel slice is completely dissolved!

**2. Bind DNA**
- Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 mL)
and load up to 700 μL sample.
-  Centrifuge for 30 s at 11,000 x g.
-  Discard flow-through and place the column back into the collection tube.
-  Load remaining sample if necessary and repeat the centrifugation step.

**3. Wash silica membrane**
-  Add 700 μL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up Column.
-  Centrifuge for 30 s at 11,000 x g.
-  Discard flow-through and place the column back into the collection tube.

**4. Dry silica membrane**
- Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely.
Make sure the spin column does not come in contact with the flow-through while
removing it from the centrifuge and the collection tube.

**5. Elute DNA**
-Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 mL
microcentrifuge tube (not provided).
-Add 15–30 μL Buffer NE and incubate at room temperature (18–25 °C) for 1 min.
Centrifuge for 1 min at 11,000 x g.

Gel extraction
**1. Bind DNA**
- Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 mL)
and load up to 700 μL sample.
-  Centrifuge for 30 s at 11,000 x g.
-  Discard flow-through and place the column back into the collection tube.
-  Load remaining sample if necessary and repeat the centrifugation step.

**2. Wash silica membrane**
-  Add 700 μL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up Column.
-  Centrifuge for 30 s at 11,000 x g.
-  Discard flow-through and place the column back into the collection tube.

**3.** Dry silica membrane
- Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely.
Make sure the spin column does not come in contact with the flow-through while
removing it from the centrifuge and the collection tube.

**4.** Elute DNA
-Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 mL
microcentrifuge tube (not provided).
-Add 15–30 μL Buffer NE and incubate at room temperature (18–25 °C) for 1 min.
Centrifuge for 1 min at 11,000 x g.

Miniprep – Plasmid Isolation (adapted from Macherey-Nagel) Protocol

1. **Cultivate and harvest bacterial cells**
Use **2 mL** of each LB culture of the previous day (pFliC L0), pellet cells in a
standard benchtop microcentrifuge for **30s at 11, 000 x g**. Discard the
supernatant and remove as much of the liquid as possible. Repeat this step until
there is 1ml left in the previously 5ml LB culture.

2. **Cell lysis**

**1.** Add **250 μL Buffer A1.** Resuspend the cell pellet completely by vortexing.
**Make sure no cell clumps remain before addition of Buffer A2!**

**2.** Add **250 μL Buffer A2.** Mix gently by inverting the tube 6-8 times. Incubate at
room temperature for **up to 5 min**> or until lysate appears clear.
**3.** Add **300 μL Buffer A3.**> Mix thoroughly by inverting the tube 6-8 times **until
blue samples turn colorless completely!**

3. **Clarification of lysate**

**4.** Centrifuge for **10 min at 15,000 x g** at room temperature.

4. **Bind DNA**

**5.** Place a Nucleospin Plasmid Column in a Collection Tube (2 mL) and decant
the supernatant from step III or pipette a **of 750 μL** of the supernatant onto
the column. Centrifuge for **1 min at 11,000 x g**. Discard flowthrough and
place the Nucleospin Plasmid Column back into the collection tube.
**Repeat this step to load the remaining lysate.**

5. **Wash silica membrane**

**6.** Add **500 μL Buffer AW**, and centrifuge for **1 min at 11,000 x g** before
proceeding with Buffer A4.

**7.** Add **600 μL Buffer A4** (supplemented with ethanol) Centrifuge for 1 min at
11,000 x g. Discard flowthrough and place the Nucleospin Plasmid Column
back into the **empty**> collection tube.

6. **Dry silica membrane**

**8.** Centrifuge for **3 min at 11,000 x g** and discard the collection tube.

7. **Elute DNA**

**9.** Place the Nucleospin Plasmid Column in a 1.5 mL microcentrifuge tube and
add 40 μL Buffer AE.

**10.** Centrifuge for **1 min at 11,000 x g**.

Tyrosine experiments

The previous day you have done a transformation to have fresh bacteria 2 plates:
one plate BL21 with tyrosinase and one BL21 without plasmid
Next day in the morning you pick 4 colonies and make liquid cultures LB 10 ml each.
3 colonies from the Tyrosinase an 1 colony from the NO plasmid
Incubate at 210 rpm 37 o C (approximately 4 hours) In order to get OD600=0,8
Negative Controls:
● Tyrosinase Buffer without Bacteria
● Tyrosinase Buffer and BL21 bacteria with no insert
● Bacteria with Tris HCl CuSO4

Each Negative control for the 3 colonies
Make 1 ml aliquotes in 1,5 ml tubes
Centrifuge 3000 g 15 mins take with a pipette the supernatant and resuspend in 1
ml Tyrosinase Buffer(50mM Tris HCl, 10μM CuSO4, 1g/L L-Tyrosine)
Incubate at 30 o C static conditions for 6 hours.
After 6 Hours 100ul from each aliquote culture is taken to check if OD600 is still 0,8
Centrifuge 2500g for 15 mins Then take from each aliquote 3 x 100 ul (3 wells) from
the supernatant to measure at 400nm

2. **Cell lysis**

**1.** Add **250 μL Buffer A1.** Resuspend the cell pellet completely by vortexing.
**Make sure no cell clumps remain before addition of Buffer A2!**

**2.** Add **250 μL Buffer A2.** Mix gently by inverting the tube 6-8 times. Incubate at
room temperature for **up to 5 min**> or until lysate appears clear.
**3.** Add **300 μL Buffer A3.**> Mix thoroughly by inverting the tube 6-8 times **until
blue samples turn colorless completely!**

3. **Clarification of lysate**

**4.** Centrifuge for **10 min at 15,000 x g** at room temperature.

Plate Reader Assay

**Settings optimized for EGFP based assays in 96 well plates:**

Excitation: 485 nm
Excitation Bandwidth: 9nm
Emission: 515 nm
Emission Bandwidth: 20 nm
Integration time: 20 us
Gain: Optimal
Number of Flashes: 10 Bottom optics

**Controls :**

**Control 1: Untransformed cells**
This gives us the background fluorescence output which is calculated by growing
cells without EGFP (EGFP-less) in the same medium and microplate. The measured
EGFP-less fluorescence is subtracted from the final fluorescence output of
transformed cells (with EGFP).
**Control 2 : Empty walls**
This gives the absorbance of plate itself . This measure is going to subtract from the
final measurement of our construct.
**Control 3 : Walls with medium and antibiotic (if it is necessary )**
M9 medium with antibiotic will give us the background fluorescence output. M9
mediun it does emit fluorescence and is transparent , but the addition of the
antibiotic may give a false absorbance.

**1.**> Previous day : complete the transformation with the desired DNA .
**2.** Pick 3 colonies from each plate.
**3.** Put each colony into 5mL of LB pre-cultures and incubate O/N , 37 o C and 210
rpm ( 3 colonies from agar plate 3 liquid colonies)
**4.** Next morning , measure the OD600 of the overnight cultures.
**5.** For each one of the colonies : Dilute the culture to target OD600=0,1 in 5mL
of desired medium and antibiotic. The number of M9 cultures depends on
the timepoints you are going to choose.
(In our protocol we are going to measure the OD600 and F1: 0h ,1h,2h, 4h,8h,
10h,24h.
Ιn total : 7 cultures of 5mL of medium. )
So for one colony : take 35mL of M9 medium and desired antibiotic and
dilute the pre-culture to target OD600=0,1.
Then take 7 sterilized eppendorfs and deposit 5mL of the initial liquid culture
( M9 +antibiotic)
**6.** Incubate each culture : 37 o C and 210 rpm . The duration of incubation varies
for each liquid culture.
(By the end of this step : 1 construct 3 colonies from the agar plate each
colony into one unique liquid culture of M9 medium and desired antibiotic
M9 liquid colony: distribute into 7 new cultures (5mL each tube )each one
represents a different timepoint for measure.)
**7.** In due time : take 600 μl of each liquid culture and split into 3 walls ( 200μl a
walls , technical replicates )
**8.** Set the optimized parameters and measure.

Colony PCR

**1.** Mark the colonies on the plate and label them with numbers If you
have a negative control plate, mark 1 to 3 colonies
**2.** Take out PCR tubes and label them as the colonies
**3.** Be sure that you have prepared replica plates and draw to the back
of
them distinct sections; one per colony. Label them as the above
**4.** Prepare the MasterMix
Number of reactions: number of colonies, 1 NTC without colony,
1 to 2 NTC with the colony/ies from the NTC plate, 1extra/10
colonies
**5.** Transfer 25 ul MM to the PCR tubes
**6.** Pick a colony with a tip
**7.** ‘’Poke”softlytothereplicaplate
**8.** DissolvetherestinthePCRtube
**9.** Repeat for all the colonies

**10.** Quick spin down

|  |  |
| --- | --- |
| Reagents | Volumes (ul) |
| PCR water | 19,9 |
| 10x Kapa Taq buffer A | 2,5 |
| 10μM dNTPs | 0,5 |
| 10μΜ Forward Primer F2 | 1 |
| 10μΜ Reverse Primer F2 | 1 |
| Kapa Taq Polymerase (5u/μl) (Kapa Biosystems) | 0,1 |

● See the following protocol on a thermocycler

|  |  |  |
| --- | --- | --- |
| Temperature | Time | Cycles |
| 95°C | 3' | 1 |
| 95°C | 30'' | 35 |
| Primer’s Tm - 5°C | 30'' | 35 |
| 72 °C | 0,06sec/bp | 35 |
| 72 °C | 0,06sec/bp | 1 |
| 4°C | ∞ | ∞ |

Run the PCR product on an agarose gel and check the result
Pick the right colony/ies from the plate and prepare liquid cultures for
minipreps

Go Back

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#### Contact

* igemthessaly@gmail.com

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# Team:Thessaly/Results

Team: Thessaly - 2020.igem.org

[![alternative](https://static.igem.org/mediawiki/2020/d/d5/T--Thessaly--Transparent-callig.jpg)](https://2020.igem.org/Team%3AThessaly)

* [HOME(current)](https://2020.igem.org/Team%3AThessaly)
* ABOUT

  [OUR TEAM](https://2020.igem.org/Team%3AThessaly/Team)[ATTRIBUTIONS](https://2020.igem.org/Team%3AThessaly/Attributions)
* PROJECT

  [PROJECT DESIGN](https://2020.igem.org/Team%3AThessaly/Design)[PROJECT DESCRIPTION](https://2020.igem.org/Team%3AThessaly/Description)[EXPERIMENTS](https://2020.igem.org/Team%3AThessaly/Experiments)[PROOF OF CONCEPT](https://2020.igem.org/Team%3AThessaly/Proof_Of_Concept)[ENGINEERING](https://2020.igem.org/Team%3AThessaly/Engineering)[MODEL](https://2020.igem.org/Team%3AThessaly/Model)[NOTEBOOK](https://2020.igem.org/Team%3AThessaly/Notebook)[RESULTS](https://2020.igem.org/Team%3AThessaly/Results)
* PARTS

  [PARTS OVERVIEW](https://2020.igem.org/Team%3AThessaly/Parts)[PARTS COLLECTION](https://2020.igem.org/Team%3AThessaly/Part_Collection)[CHARACTERIZATION](https://2020.igem.org/Team%3AThessaly/Contribution)[MEASUREMENTS](https://2020.igem.org/Team%3AThessaly/Measurement)
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* IMPLEMENTATION

  [IMPLEMENTATION OVERVIEW](https://2020.igem.org/Team%3AThessaly/Implementation)[SAFETY](https://2020.igem.org/Team%3AThessaly/Safety)
* [MEDAL CRITERIA(current)](https://2020.igem.org/Team%3AThessaly/medal-crit)
* [GIANT JAMBOREE(current)](https://2020.igem.org/Team%3AThessaly/jamboree/)

# Results

#### Overview

Because of the unique situation this year, our access in the lab was limited. However, we started building our designed modules to lay the groundwork for future experiments. To accomplish that, we repurposed the GoldenBraid modular assembly strategy to enable bacterial expression of multigenic constructs, utilizing the SEVA standardized plasmid collection. We then assembled the GPCR-Tango Module, the Tet-off Module, the Prom Module and the Tyrosine-Tyrosinase Reporter Module that comprise the Monitoring System of Amalthea. Finally, we characterized and tested both Prom Module and Tyrosine-Tyrosinase Module, to prove that our system works.

#### Strategy

Our experiments were designed in a way that we could build new genetic Modules combining basic DNA parts in a standardized way.  In order to increase the multigene assembly efficiency and perform successfully our experiments following the GoldenBraid assembly strategy that is used for Plant Synthetic Biology. This strategy is based on Type IIS restriction enzymes allowing the creation of our genetic circuits or our Modules. We modified our basic DNA parts designing compatible sites, in order to combine them and create final constructs that consist of a promoter, a CDS, and a terminator and our final Modules with this repeated pattern. In that way, we built an increasingly complex multigene System that can facilitate the heterologous expression of proteins in bacteria.

#### Level 0 cloning

The iGEM parts are the legacy of every Team for the future iGEM Teams. Using the iGEM registry is the best way to build your experimental design. This year we submitted several interchangeable Level 0 parts based on the TypeIIS Assembly. We include both project-specific and general parts, which are compatible for GoldenBraid and MoClo. This way, we make a contribution for future iGEM Teams.

#### Promoters

Click here to read more.

[BBa\_K3505012](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505012)

![alternative](https://static.igem.org/mediawiki/2020/6/6a/T--Thessaly--results-01.png)

**Figure1.** (U=Uncut , C= Cut) Restriction digestion of AndersonJ23115 with : EcoRI (C1a & C2a),Expected bands : 2156 bp, XhoI (C1b & C2b),Expected bands : 1264 + 892 bp Positive result: The set of C1a and C1b (same sample).

[BBa\_K3505014](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505014)

![alternative](https://static.igem.org/mediawiki/2020/1/17/T--Thessaly--results-02.png)

**Figure 2.** (U=Uncut , C= Cut) Restriction digestion of TetO (C1-C4 ) with : Not-HF(C1-C4) , Expected bands : 2046 + 129 bp ,Positive result: C1,C2,C4

[BBa\_K3505013](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505013)

![alternative](https://static.igem.org/mediawiki/2020/7/7f/T--Thessaly--results-03.png)

**Figure 3.** (U=Uncut , C= Cut)Restriction digestion of LacO (C3 & C4) with : EcoRV + HindIII (C3 & C4) , Expected bands : 2173bp ,Positive result : C3 + C4

[BBa\_K2924016](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2924016)

![alternative](https://static.igem.org/mediawiki/2020/b/b6/T--Thessaly--results-04.png)

**Figure 4.** Restriction digestion of pFlic (C1-C6) with : with SlaI, Expected bands: 1314 bp + 892 bp, and EcoRI, Expected bands: 2206 bp. Positive result : C5 + C6.

[BBa\_K3505015](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505015)

![alternative](https://static.igem.org/mediawiki/2020/c/cb/T--Thessaly--results-05.png)

**Figure 5.** (U=Uncut , C= Cut) Restriction digestion of PrpB (C1 & C2) with : EcoRV + HindIII (C1 & C2) , Expected bands : 2297bp ,Positive result: C1 & C2

[BBa\_K3505016](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505016)

![alternative](https://static.igem.org/mediawiki/2020/b/bc/T--Thessaly--results-06v2.png)

**Figure 6.** (U=Uncut C=Cut) Restriction Digestion of araC:ParaBAD with EcoRI+ PstI ,Expected bands: 2029+1996 , Positive result

#### Coding secuences

Click here to read more.

[BBa\_K3505003](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505003)

![alternative](https://static.igem.org/mediawiki/2020/f/fb/T--Thessaly--results-07.png)

**Figure 7.** (U=Uncut C=Cut) Restriction Digestion of LacI with EcoRI + SlaI, Expected bands : 3189 +2300 + 892 bp, Positive Result:C2

[BBa\_K3505005](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505005)

![alternative](https://static.igem.org/mediawiki/2020/5/57/T--Thessaly--results-08.png)

**Figure 8.** (U=Uncut C=Cut) Restriction digestion of TetR (C2 -C4) with : SpeI + EcoRI (C2 - C4) , Expected bands : 2047 + 680 bp Positive result : C4

[BBa\_K3505021](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505021)

[BBa\_K3505022](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505022)

![alternative](https://static.igem.org/mediawiki/2020/2/28/T--Thessaly--results-09.png)

**Figure 9.** (U=Uncut C=Cut) Restriction digestion of RraA (C2 ) with : PstI + EcoRI , Expected bands: 2029, 564 bp
Restriction digestion of DjlA (C4 )with EcoRI, PstI Expected bands 2029, 894 bp

[BBa\_K3505018](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505018)

![alternative](https://static.igem.org/mediawiki/2020/2/21/T--Thessaly--results-10.png)

**Figure 10.** (U=Uncut C=Cut) Restriction digestion of sfGFP (C4 ) with : EcoRI, PstI Expected bands 2029, 794

[BBa\_K3505020](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505020)

![alternative](https://static.igem.org/mediawiki/2020/8/80/T--Thessaly--results-11.png)

****Figure 11.** (U=Uncut C=Cut) Restriction digestion of ECFP (C1-C3 ) with : SlaI, Expected bands : 1934 +892 bp, Positive Result : C1,C3**

[BBa\_K3505019](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505019)

![alternative](https://static.igem.org/mediawiki/2020/a/a9/T--Thessaly--results-12.png)

**Figure 12.** (U=Uncut C=Cut) Restriction digestion of EGFP (C1-C4 ) with : PvuII, Expected bands : 2826 bp , Positive Result : C4

[BBa\_K3505006](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505006)

![alternative](https://static.igem.org/mediawiki/2020/9/92/T--Thessaly--results-13.png)

**Figure 13.** (U=Uncut C=Cut) Restriction digestion of Tyr-Aida (C1-C4 ) with : EcoRV, EcoRI, BamHI. Expected bands 1378, 896, 700, 550, 490, 430, 200. Positive result : C

[BBa\_K3505025](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505025)

![alternative](https://static.igem.org/mediawiki/2020/9/9f/T--Thessaly--results-14.png)

**Figure 14.** (U=Uncut C=Cut) Restriction Digestion of b-arrestin-TEV protease with SlaI, Expected bands : 2014 bp + 892 bp + 760 bp + 394 bp, Positive Result: green arrows

[BBa\_K3505023](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505023)

![alternative](https://static.igem.org/mediawiki/2020/f/f1/T--Thessaly--results-15.png)

**Figure 15.** (U=Uncut C=Cut) Restriction Digestion of FFAR2:V2tail:TCS with BamHI, Expected bands : 3204bp, Positive Result:C2,C3

[BBa\_K3505017](http://parts.igem.org/Part%3ABBa_K3505017)

![alternative](https://static.igem.org/mediawiki/2020/e/e2/T--Thessaly--results-16.png)

**Figure 16.** (U=Uncut C=Cut) Restriction digestion of Terminator (C3 )with : EcoRI & PstI ( C3 ), Expected bands : 2029 + 209 bp , Positive result : C3

#### Vectors

To facilitate standardized and modular cloning in our experiments. We built four standardized vectors, which are a bacterial-expression variation of the GoldenBraid standard (Sarrion-Perdigones et al. 2013), combined with SEVA plasmids. Using our system, the whole SEVA collection can be utilized to create vectors with a desirable of antibiotic resistance genes and ORIs by targeting the T0 and T1 terminators that are universal.  [Explore our full design here](https://2020.igem.org/Team%3AThessaly/Engineering)

##### Assembly of bacterial GoldenBraid vectors

pSEVAb23 (Damalas et al., 2020) was used as a backbone for the pGBD1 alpha 1R and pGBD1 alpha 2 using Q5 (NEB) PCR amplification because Level cloning has kanamycin resistance. Resulting in a backbone with kanamycin and BsmBI BsaI sites. The backbones were amplified with the primers from Table 1. Then Agarose Gel Electrophoresis (AGE) is conducted. And after that Gel extraction and Clean up isolating the backbones with the overhangs.

Then with BsmBI restriction digestion, the LacZa module was taken from the pDGB1 alpha vectors and with BsaI from the pDGB1 omega vectors. The whole reactions were loaded and AGE was conducted.  Then Gel extraction and Clean up isolating the LacZa insert.

The same for pSEVAb43 that was used as the backbone for the pGBD1 omega 1R and pGBD1 omega 2 using PCR amplification because Level cloning has spectinomycin resistance. Resulting in a backbone with spectinomycin and BsmBI BsaI sites.

Digestion-Ligation with BsmBI and T4 Ligase (NEB for GB cloning) Backbones and Inserts were combined resulting in our *E. coli* friendly Plasmids (Figures 17, 18).

|  |  |
| --- | --- |
| CPrimer | Sequence from 5’ to 3’ |
| Alpha 1R FORWARD | ATctcctgagacggatccagtaggacaaatccgccgcc |
| Alpha 1R REVERSE | ATgtcatgagacggatccttgttcagaacgctcggttgcc |
| Alpha 2 FORWARD | ATtgactgagacgaagcttagtaggacaaatccgccgcc |
| Alpha 2 REVERSE | ATcgcttgagacgaagcttttgttcagaacgctcggttgcc |
| Omega 2 FORWARD | ATtgactgagaccgatatcagtaggacaaatccgccgcc |
| Omega 2 REVERSE | ATcgcttgagaccgatatcttgttcagaacgctcggttgcc |
| Omega 1 FORWARD | Tgcggccgcggatatattgtggtgtaacg |
| Omega 1 REVERSE | Tgcggccgcccaatatatcctgtcaggat |

![alternative](https://static.igem.org/mediawiki/2020/3/34/T--Thessaly--results-17.png)

![alternative](https://static.igem.org/mediawiki/2020/f/f5/T--Thessaly--results-17-2.png)

**Figure 17.** **A)** pDGB1 alpha 1R vector with SEVA23 backbone **B)** pDGB1 alpha 2 vector with SEVA23 backbone.

![alternative](https://static.igem.org/mediawiki/2020/1/1a/T--Thessaly--results-18.png)

![alternative](https://static.igem.org/mediawiki/2020/4/4d/T--Thessaly--results-18-2.png)

**Figure 18. A)** pDGB1 omega 2 vector with SEVA43 backbone **B)** pDGB1 omega 1R vector with SEVA43 backbone.

##### Verification of our constructs

As it is showed at Figure 17 and 18B the 3 vectors pDGB1 alpha 1R, pDGB1 alpha 2, pDGB1 omega 2 were correct.

![alternative](https://static.igem.org/mediawiki/2020/6/66/T--Thessaly--results-19.png)

**Figure 19.** pDGB1 a1R digested with BamHI.
Expected bands: 2847 bp + 385 bp + 239 bp.
pDGB1
a2 digested with HindIII. Expected bands: 2847 bp + 416 bp + 206 bp.

![alternative](https://static.igem.org/mediawiki/2020/7/71/T--Thessaly--results-20.png)

![alternative](https://static.igem.org/mediawiki/2020/4/4f/T--Thessaly--results-20-2.png)

**Figure 20. A)** pDGB1 omega1R digested with BamHI. Expected bands: 3153 bp + 382 bp + 239 bp. **B)** pDGB1 omega2 digested with EcoRV and HindIII. Expected bands: 2900 bp + 398 bp + 223 bp.

But the vector pDGB1 omega 1R had a problem on its primers’ set so the reverse idea was conducted. We designed a new set of primers having overhangs with NotI (Table 1). We amplified the LacZa module including the BsmBI BsaI sites from the original pDGB1 omega 1 vector. Then we digested with NotI the pSEVA43, in order to take the backbone.

At the beginning we had designed primers for pDGB1 omega 1, but cutting with NotI the LacZa module with the BsmBI and BsaI sites could be ligated in either orientation. Through sequencing we confirmed that it was ligated with reverse orientation (Figure 20 A). So omega 1R is our final vector. This result has no problem with the cloning though. In the future, we will replace our current ORI (pBBR1), which provides medium/low copy numbers, with a high copy one to facilitate cloning of complex constructs. This is easy, because according to the SEVA standard, we will digest with rare restriction enzymes PshAI and SwaI.

#### Tango-GPCR Module

The potential of SCFAs as diagnostic and therapeutic biomarkers can be utilized, by engineering a GPCR-based artificial signaling pathways in *E. coli*.This synthetic signaling pathway is based on the TANGO high-throughput screening technology, that takes advantage of the b-arrestin capabilities to bind to the GPCR receptor. [For the whole design click here.](https://2020.igem.org/Team%3AThessaly/Engineering)

##### Assembly of Tango-GPCR Module

The Tango module consits of 4 TUs. The first is an arabinose inducible synthetic FFAR linked with LacI as seen at (Figure 21)
based upon the work done by (Barnea et al. 2008)
The second TU is a fusion of the b-arrestin-2 with the TEV protease, which liberates the LacI from the GPCR bioreceptor as seen at (Figure 22)
The third TU is a Lac regulated Expression of eCFP see(Figure 23)
The fourth TU is an arabinose inducible antitoxic proteins seen at (Figure 24)
We have verified the correct assembly both by diagnostic digestions and Sanger Sequencing. Our goal is to test this system for iGEM 2021.

##### Verification of Tango-GPCR Module

[BBa\_K3505025](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505025)

![alternative](https://static.igem.org/mediawiki/2020/3/35/T--Thessaly--results-21.png)

**Figure 21.** (U=Uncut , C= Cut) Restriction digestion a1R:ParaBAD:RBS-FFAR2:V2tail:TCS-Lac-Double terminator (C1a-C4b) with : BamHI(C1a-C4a) , Expected bands : 2847+2225 bp , EcoRV (C2a-C2b) ,Expected bands : 3587 bp + 2845 bp, Positive result: C1,C2,C3,C3 (C1a and C1b is the same sample etc)

[BBa\_K3505026](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505026)

![alternative](https://static.igem.org/mediawiki/2020/9/93/T--Thessaly--results-22.png)

**Figure 22.** (U=Uncut , C= Cut) Restriction digestion of ParaBAD:RBS:B-arrestin -TEV protease -Double Terminator (C1-C 4), with :HindIII (C1a-C4a), Expected bands : 2847+2490+719 bp , EcoRV (C1b-C4b) , Expected bands:3988 bp+2214 bp. Positive result: C1,C2,C3,C4. (C1a and C1b -same sample etc)

[BBa\_K3505030](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505030)

![alternative](https://static.igem.org/mediawiki/2020/4/4f/T--Thessaly--results-23.png)

**Figure 23.** (U=Uncut , C= Cut) Restriction digestion of AndersonJ23115:LacO -ECFP -Double Terminator (C1-C 4), with : BamHI (C1-C4) , Expected bands:2847 +952 bp. Positive result: C1,C2,C3,C4.

[BBa\_K3505039](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505039)

[BBa\_K3505038](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505038)

![alternative](https://static.igem.org/mediawiki/2020/d/dd/T--Thessaly--results-25.png)

**Figure 24.** Restriction Digestion of ParaBAD-DjlA-Terminator (C1-C2) with EcorV and BamHI ,Expected bands 4212+ 853 bp, Positive Result : C1
**Restriction Digestion of** ParaBAD-RraA-Terminator (C3-C4) with EcoRV Expected bands 3471+1264 bp , Positive Result C4

#### Tet-off Module

Our aim was to reduce the complexity of bacterial GPCR expression and we designed a simple NOT-GATE system induced by anhydrotetracycline (aTC). Behind this module there is the same logical structure with the GPCR -Tango Assay. Α modular system that can simulate the detection of absence of SCFAs from the GPCR.
Tetracycline Repressor (TetR) is a well-characterized transcriptional factor and we can make use of a tetracycline derivate, anhydrotetracycline (aTC) a strong chemical repressor (Gossen and Bujard, 1993). For full design click here.

##### Assembly of Tet-off Module

This module consists of several individual Level α transcriptional units (TUs). In order to create the NOT-GATE device, we assembled a constitutive expression TetR TU as seen at (Figure 26), a LacI-regulated ECFP TU AndersonJ23115:lacO-eCFP-double terminator (Figure 23) , and a tet-regulated expression of Lac inhibitor.

##### Verification of Tet-off Module

[BBa\_K3505044](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505044)

![alternative](https://static.igem.org/mediawiki/2020/5/5e/T--Thessaly--results-25-6.png)

**Figure 25.** (U=Uncut , C= Cut) Restriction Digestion of TE: AndersonJ23115:TetO-EGFP-double terminator with EcoRV(C1-C2). Expected bands : 2847+ 954 bp. Positive result: C1,C2.

[BBa\_K3505005](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505005)

![alternative](https://static.igem.org/mediawiki/2020/f/fb/T--Thessaly--results-27.png)

**Figure 26.** (U=Uncut , C= Cut) Restriction Digestion of T: AndersonJ23115-TetR-double terminator digested with HindIII (C3-C4). Expected bands : 2847 + 836 bp. Positive result : C3,C4.

[BBa\_K3505034](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505034)

![alternative](https://static.igem.org/mediawiki/2020/0/0d/T--Thessaly--results-28.png)

**Figure 27.** (U=Uncut C=Cut) Restriction digestion of TL: AndersonJ23115:Tet0-LacI-double terminator(C5-C8 ) with: HindIII + NheI (C5-C18) , Expected bands : 2573+891+427,Positive result : C5,C6

After level α constructs were completed , and more specifically the AndersonJ23115:LacO-eGFP-double terminator (Figure 23) and AndersonJ23115:TetO-eGFP-double terminator (Figure 24)
, we started plate-reader assays before sequencing results arrived. This construct is crucial for our experiments as it used to the majority of our modules.
After sevreal measurements that yielded no fluorescence, we came to a conclusion that something was wrong with this transcription unit as no detectable fluorescence was produced in our initial experiments testing the eGFP.
After the sequencing result came in , we noticed single point mutations in our eGFP clones. After we BLASTed the sequences, eCFP was 100% identical with the sequencing sample.It turns out that due to (most likely) wrong starting template, we had cloned eCFP instead of eGFP, which was the reason for non-detectable fluorescence. The next step was to adapt our protocol to new standards and create a new protocol.

Measurements are the average of 9 total replicates (3 biological replicates and 3 technical replicates per biological replicate). Error bars represent standard deviation of biological replicates.
The results of the assay can be seen in the figures below:

![alternative](https://static.igem.org/mediawiki/2020/a/a4/T--Thessaly--results-28-9.png)

**Figure 28.** Validation that J23115-TetO and J23115-LacO fusion promoters are able to drive expression of ECFP.Expression of ECFP, after 16h of incubation at 37oC , using M9 Medium.

![alternative](https://static.igem.org/mediawiki/2020/c/cb/T--Thessaly--results-29.png)

**Figure 29.** Bacterial Growth after 16h incubation at 37 oC with M9 medium.

#### Prom Module

We designed a a promoter-based module for SCFAs detection that performs as a NOT-GATE devices, in case the GCPR-Tango module is not functional. We used SCFA-induced promoters, pFliC (Tobe, Nakanishi and Sugimoto, 2010) and prpBCDE (Tsang, Horswill and Escalante-Semerena, 1998), so as two SCFAs work as an input. Downstream of them is a transcriptional factor, LacI, which binds to a Lac Operator, when is activated, leading to the inhibition of the expression of the reporter gene. [Explore our full design here](https://2020.igem.org/Team%3AThessaly/Engineering)

##### Assembly of Prom Module

Our goal was to create our final constructs that will consist of our promoter, LacI, LacO and the reporter gene, in order to create a NOT-GATE. To do so we created different constructs for the expression of LacI and the expression of our reporter gene, eCFP. The expression of LacI was regulated by our SCFAs-induced promoters as you can see at (Figures 30, 31), while our reporter gene, eCFP was downstream of Lac Operator (LacO)(Figure 23).These constructs were combined into a final omega1R as seen at Figure 36, creating a NOT-GATE and allowing us to test our Module. [For more information about our Proof of Concept here](https://2020.igem.org/Team%3AThessaly/Proof_Of_Concept)

##### Verification of Prom Module

[BBa\_K3505031](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505031)

![alternative](https://static.igem.org/mediawiki/2020/2/2a/T--Thessaly--results-30.png)

**Figure 30.** Restriction Digestion of LEVEL 1: prpB-lacI-terminator with HpaI (C1-C2). Expected band: 4286 bp. Positive result : C1 .

[BBa\_K3505027](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505027)

![alternative](https://static.igem.org/mediawiki/2020/6/67/T--Thessaly--results-31.png)

**Figure 31.** Restriction Digestion of LEVEL 1: pFliC-LacI-terminator digested with BamHI (C1), Expected bands: 2847 bp + 1348 bp, and with HpaI (C4) , Expected bands 4195 bp. Positive result : C1 +C4

We created Level A constructs that consisted of our promoter, pFlic or prpBCDE, LacI and terminator into an alpha1R vector and a constitutive promoter of iGEM Registry, Anderson J23115, combined with the Lac Operator (LacO), eCFP and terminator into alpha2 vector.(Figure 23)

[BBa\_K3505036](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505036)

![alternative](https://static.igem.org/mediawiki/2020/2/26/T--Thessaly--results-32.png)

**Figure 32.** Restriction Digestion of omega 1R pFliC:RBS-LacI-Terminator-pAndersonJ23115:LacO:RBS-ECFP-terminator with EcoRV+ HindIII (C1-C3). Expected bands: 3734 + 1699 bp. Positive result : C2.

Then Level A constructs were combined together, into omega1R vector creating our final constructs and our Prom Module regarding pFliC promoter that we further characterized for iGEM. [For more information about our Characterization here](https://2020.igem.org/Team%3AThessaly/Contribution)

Then we transformed this construct into MC1061 bacteria strains, as they are our go-to stains for protein expression of our reporter gene. Our goal was accomplished and then we had to test the function of our NOT-GATE. We added SCFAs based on the gathered data from our Characterization [Read more](https://2020.igem.org/Team%3AThessaly/Contribution), modeling [Read more](https://2020.igem.org/Team%3AThessaly/Model) and observed the decrease of the expression of eCFP at different time-points see (Figures 33-36).

![alternative](https://static.igem.org/mediawiki/2020/f/f4/T--Thessaly--results-33.png)

**Figure 33.** NOT-GATE-regulated ECFP fluorescence after in the presence or absence of 2mM acetate.

![alternative](https://static.igem.org/mediawiki/2020/9/9a/T--Thessaly--results-34.png)

**Figure 34.** Cell growth in the presence or absence of 2mM acetate.

![alternative](https://static.igem.org/mediawiki/2020/b/b7/T--Thessaly--results-35.png)

**Figure 35.** NOT-GATE-regulated ECFP fluorescence after in the presence or absence of 2mM propionate.

![alternative](https://static.igem.org/mediawiki/2020/1/1e/T--Thessaly--results-36.png)

**Figure 36.** Cell growth in the presence or absence of 2mM propionate.

The results show that there is a noticeable difference regarding the expression of eCFP, when we add 2mM acetate or propionate. We hypothesize that the gap will be bigger when adding butyrate, which is the main inducer for pFliC, and which we didn’t have in the lab as per Wiki Freeze date. However, our data indicate that changing the conditions, adding acids, affect our Module in the way we expected as it acts as a NOT-GATE circuit.

[For more information click here](https://2020.igem.org/Team%3AThessaly/Proof_Of_Concept)

#### Reporter Module

Tyrosine-Tyrosinase is a reporter module that has the ability of converting the presence of L-Tyrosine to an electrochemical signal for convenient readout. For extensive experimental design click here.

[For extensive experimental design click here.](https://2020.igem.org/Team%3AThessaly/Engineering)

##### Assembly of Reporter Module

Tyrosinase has the ability of converting L-Tyrosine to L-Dopa and L-Dopa quinone which is electrochemically detectable (Figure37). VanArsdale expressed and measured a Tyrosine fused with an autotransporter for membrane transportation under control of an inducible promoter(VanArsdale et al., 2020).

We constructed a constitutive expression of Tyrosinase as seen at Figure 41, expecting Tyrosinase to stay on the membrane and wait for L-Tyrosine to appear.

##### Verification of Reporter Module

[BBa\_K3505035](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3505035)

![alternative](https://static.igem.org/mediawiki/2020/1/11/T--Thessaly--results-37.png)

**Figure 37.** Restriction Digestion of pAndersonJ23115:RBS-tyrosinase:AIDA-terminator  with BamHI(C1-C3). Expected bands 2847+1883+520+353 bp. Positive result : all.

We transformed this construct into MC1061 expression bacteria and conducted a Tyrosinase assay as seen in (Figure 38). We added exogenous L-Tyrosine after the expression of our protein, while included appropriate controls to obtain a specific signal. The measurements resulted in no detectable output, as the same measurements were captured either from Bacteria that had the Tyrosinase or no proper Insert, as seen at (Figure 38).

We hypothesize that constitutive expression of the proteins leads to toxicity and subsequent impaired cell growth, as seen in (Figure 39). Notably, several biological replicates of our insert grew at half the speed compared an empty vector control.In our next Design-Built-Test cycle, we will include an Arabinose inducible promoter to replace the constitutive one. The choice of the specific promoters has two main reasons: 1) We already have a verified clone in the lab, so using out modular assembly system we can just replace the promoters, and 2) Our goal is to overexpress Tyrosinase along with GPCR expression in our GPCR-Tango Module, to synchronize SCFA detection and signal production. Inducing by the same promoter can provide this feature.

![alternative](https://static.igem.org/mediawiki/2020/4/44/T--Thessaly--results-38.png)

**Figure 38.** Testing the activity of Tyrosinase measuring the formation of melanin after the conversion of L-Tyrosine to L-dopa.

![alternative](https://static.igem.org/mediawiki/2020/4/44/T--Thessaly--results-38.png)

**Figure 39.** Bacterial Growth after 6h at 30oC incubation with Tyrosinase Assay Buffer.

#### References

Alejandro Sarrion-Perdigones, Marta Vazquez-Vilar, Jorge Palací, Bas Castelijns, Javier Forment, Peio Ziarsolo, José Blanca, Antonio Granell, Diego Orzaez (2013). “GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology.” *Plant Physiology* , 162 (3) 1618-1631; DOI: 10.1104/pp.113.217661

Barnea, G., Strapps, W., Herrada, G., Berman, Y., Ong, J., Kloss, B., Axel, R., & Lee, K. J. (2008). The genetic design of signaling cascades to record receptor activation. *Proceedings of the National Academy of Sciences of the United States of America*, 105(1), 64–69. https://doi.org/10.1073/pnas.0710487105

Damalas, S., Batianis, C., Martin‐Pascual, M., Lorenzo, V. and Martins dos Santos, V., 2020. SEVA 3.1: enabling interoperability of DNA assembly among the SEVA, BioBricks and Type IIS restriction enzyme standards. *Microbial Biotechnology*, 13(6), pp.1793-1806.

Gossen, M. and Bujard, H., 1993. Anhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells. *Nucleic Acids Research*, 21(18), pp.4411-4412.

Tobe, T., Nakanishi, N. and Sugimoto, N., 2010. Activation of Motility by Sensing Short-Chain Fatty Acids via Two Steps in a Flagellar Gene Regulatory Cascade in Enterohemorrhagic *Escherichia coli. Infection and Immunity*, 79(3), pp.1016-1024.

Tsang, A., Horswill, A. and Escalante-Semerena, J., 1998. Studies of Regulation of Expression of the Propionate (prpBCDE) Operon Provide Insights into How Salmonella typhimurium LT2 Integrates Its 1,2-Propanediol and Propionate Catabolic Pathways. *Journal of Bacteriology*, 180(24), pp.6511-6518.

VanArsdale, E., Hörnström, D., Sjöberg, G., Järbur, I., Pitzer, J., Payne, G., van Maris, A. and Bentley, W., 2020. A Coculture Based Tyrosine-Tyrosinase Electrochemical Gene Circuit for Connecting Cellular Communication with Electronic Networks. *ACS Synthetic Biology*, 9(5), pp.1117-1128.

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#### Contact

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