iGEM 2021 — Team UMA_MALAGA

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# Team:UMA MALAGA/Notebook

Wiki Team UMA

NOTEBOOK

NOTEBOOK

November 2020

Team consolidation

Creating ATG

ATG x iGEM UMA

Social Networks to build a community

ATG meets PDI and collaborations

CEC (Consejo de Estudiantes de Ciencias de la UMA)
Teachers: Miguel Ángel Medina Torres, Enrique Vigueras y Antonio Heredia
Colaboradores: Carlos Vivar, Roger Monfort ( iGEM UPV 2018),
Nerea (iGEM Estocolmo), Laura Balcells (iGEM Tolouse 2020)

December 2020

Departments division:
Wet Lab, Dry Lab y Human Practice

Meeting collaborators, researchers and volunteers

Volunteers: SECOT (Voluntariado Senior de Asesoramiento Empresarial)
Researcher: Judit Giró (ganadora del premio Dyson 2020 por
su proyecto The Blue Box ), Belén Pastor (investigadora predoctoral en el
FIVO con una beca de la AECC)
Collaborator: Carlos Vivar, Darío (divulgador de BiotecX),
Carlos Andreu (coordinador de Dry Lab UPV iGEM 2018, posibles
webinars de MatLab y Python)

Analysing previous teams in IGEM

We obtained 10,000 euros thanks to an entrepreneurship programme of our university (K-Project, Link by Uma), which values innovative projects.

We believe in compromise, self-management, proactivity, resolution, communication and honesty

January 2021

Team reestructuration

Brainstorming

Carbon sequestration on agricultural soil
Bioremediation ex-situ, bioremediation of chlorpyrifos, bioremediation in Mar Menor
Bioplastics, PHA-based synthesis with Methylobacterium extorquens
POCT, oncometabolite detection on urine
Drought tolerance in plants through viral effectors
Aptamers as nanofertilizers

Developing best ideas

Looking for new roles to add:
Computer Scientist, Community Manager, Graphic Designer

Meeting with partners (we discussed progress):

Juan Aguilar (Fine Arts teacher x UMA) and Carlos Vivar

February 2021

Meeting with Igem Madrid Team

Developping Ideas

Meeting researchers, experts, and asotiations

Talks with differtents experts of our area

Spanish Association for Conservation Agriculture Living Soils
Jan Barten (Researcher in tomato crop improvement at Bayer)

Focus our ideas to the local problems

March 2021

We discard several ideas:

Carbon sequestration
Ex-situ bioremediation, bioremediation of chlorpyrifos, bioremediation in Mar Menor
Bioplastics, synthesise PHA-based bioplastic by Methylobacterium extorquens
POCT, detection of oncometabolites in urine
Drought tolerance
Aptamers as nanofertilisers

New ideas and a new approach

Bioremediation
Olive Oil
Biodegradation of plastics
Biopaints
Use of CBD
Olive oil mill waste
Feed supplements
Surfactants

Seeking to raise awareness with a product that directly involves the user.

Meeting with Claudia (iGEM mentor)

We worked on new ideas

We discarded all ideas but:

Bioremediation
Olive Oil
Biopaints
Olive oil mill wastes
Surfactants

April 2021

Meeting with iGEM Mexico team (on surfactants) and researcher on oil residues in olive oil mills.

María José López López from UAL on OMWs

Meeting with our collaborator/coordinator Carlos Rodríguez Caso (Associate profesor Department of Molecular Biology and Biochemistry)

Meeting with the Extremadura Association of Biochemistry in Motion

In between meetings, we worked on the ideas and came up with a solution that we are very excited about:

Bringing ideas together:
Bioremediation
Used Olive Oil
Biosurfactants
Biosurfactants
Solving a local problem such as the non-reuse of cooking oil (oil bioremediation) and trying to involve the user directly with the biopaintings in the final product.

Research on the idea

Logo and name definition for iGEM

Our movement of spreading in networks has not stopped, we are known as ATG, but the idea needs another name that represents the main idea of the project, Oil Together Painting comes to mind.

Human Resources department work to adapt the idea to the user and raise awareness of the problem.

Conclusions and progress on the idea:

**Main objective:**
Use wasted oil as a carbon and energy source for our organism of choice, which we will genetically modify to produce coloured compounds, which, together with the biosurfactants (BSs) produced under these conditions as emulsifying agents, will constitute the main elements of our biopaint.
Choice of host organism: Y. lipolytica, Candida tropicalis, Bacillus subtilis, Alkaloids, Flavonoids, Carotenoids.
**Final proposal:**
Using a strain of Y. lipolytica in a culture medium with WCO as a carbon and energy source, and coupling the detoxification of the residue with the production of pigments of the carotenoid family, using Synthetic Biology techniques. Subsequently, the process effluent, rich in pigments and bioemulsifiers, will be functionalised into a biopaint of interest to the user.

Meeting with our collaborators and PDI

We present the progress of the project

Professors: Miguel Ángel Medina Torres, Enrique Vigueras, Antonio Heredia, Carlos Rodríguez Caso
Collaborators: Carlos Vivar, Nerea (iGEM Stockholm)

May 2021

Presentation of ATG and Oil Together Painting to students from the University of Malaga

Meeting with Rodrigo Ledesma,

He gave us Y. lipolytica strains, a cassette coding for the production of beta carotenoids y and empty vector

Meeting with Belén Delgado (UMA communicator)

Presential meeting Lab Team in Green Ray

Laboratory planning, including a first proposal of materials, protocols and timing.
Contact with Rodrigo to meet and discuss the ordering process
Order plasmid map to choose restriction enzymes and order from New England Biolabs (NEB).
Develop Oil Painting scheme and research oil recycling and regulatory issues.

Meeting with Carlos Rodríguez, "you must organize the laboratory very well".

June 2021

Meeting with Claudia (iGEM Ambassador)

Wet Lab face-to-face meeting at Green Ray (a space at our University dedicated to entrepreneurship).

Meeting with Carlos Rodríguez Caso

EAnalyze Biobricks y submit to the Part registry

Meeting with Manuel Bernal, he advises us:

To have as soon as possible plasmid and yeast to transform.
Know exactly nutritional and environmental requirements for Y. lipolytica.
A file in which we know exactly what we need to create the liquid medium where our yeast will grow. He assures that it will be more difficult to create a good medium than to transform the yeast.

Meetings to organise laboratory work

Carlos Rodríguez Caso stresses that we must standardise protocols.

New England Biolabs Grant and collaboration with Imperial College de Londres

We won one of the Team Impact Grant

We already have a mentor for iGEM: Beetel

The hard work starts now, we start using Notion to better organise our tasks.

We started using TikTok

Meeting with iGEM Madrid team leader

Laboratory work begins

[Laboratory Notebook](https://static.igem.org/mediawiki/2021/2/2d/T--UMA_MALAGA--notebook.pdf)

July 2021

Half of the team got infected with Covid-19

We decided not to go to the MeetUp in Greece, because of COVID-19 and money

We made a dossier to get sponsors.

We finish iGEM's external website

We started to think about what the kit will look like for the project.

We made a dossier to get sponsors.

We start preparing collaborations with the iGEM team

Synthetic biology outreach in two schools with 8-12 year olds

August 2021

Laboratory work is becoming more and more intensive

We are still looking for sponsors

We are still thinking and accepting collaborations

Taking a closer look at the iGEM competition

We study which medals we can get

Studying how our biopaint will influence the future

We started to write and organise the wiki.

September 2021

We are finishing collaborations

The iGEM Paris team proposes an online congress for the participation of companies involved in pigment production.

We continue to write and organise the wiki

Laboratory work continues

We do PodCast

We started to prepare a possible event to promote our project.

Hard work on the wiki

We attended an entrepreneurship congress, where we met our Sponsors Link by Uma and BioAzul (Congress AgriFood)

Laboratory work continues

We continue to write and organise the wiki

We started preparing final presentations of the project

Very long and laborious days before the Wiki Freeze.

CONTACT US

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# Team:UMA MALAGA/Engineering

Wiki Team UMA

ENGINEERING SUCCESS

IDEA

Using waste cooking oil (WCO) as the substrate of yeast Yarrowia lipolytica for the beta-carotene synthesis (chosen pigment for the bio-paint creation).

We need to introduce codified proteins inside Yarrowia lipolytica by crtB, crtl y carRP to develop the steps from GGPP molecule to the beta-carotene. WCO has a lot of TAGS so the FFA synthesis increments, and in the last instance, the GGPP.

For improving the initial visualization, we created what we called "Workflow databases".

WORKFLOW DATABASES

VISUALIZATION AT ITS BEST

We had in mind the implementation of a theoretical model database to achieve the
route's visualization. What you are about to see is a relational model, it helped us to
understand how the attributes will ensemble together at the lab.
"Divide and conquer the different processes to get the best possible final result"

DESIGN

METHABOLIC
PATHWAY

![](https://static.igem.org/mediawiki/2021/8/89/T--UMA_MALAGA--methabolicpathway.png)

GENETIC CONSTRUCTIONS

SOFTWARE DESIGN

We use Benchling for preparing; Promoters, RBS, Coding Sequence Genes and Terminators to make them compatible with Biobricks technique:

Adding Preffix (EcoRI and XbaI) and Suffix (SpeI and PstI) to all parts.
For Coding Sequence Genes we add a RBS for our yeast (Yarrowia Lipolytica).

![](https://static.igem.org/mediawiki/2021/a/ae/T--UMA_MALAGA--ennew.png)

GENETIC CONSTRUCTIONS

BUILDING THE IDEA

1. Enzymatic digestion and ligation of parts. Parts are cut and joined two by two.
2. Transformation. Joined parts are ensembled to a microorganism. Its job is to amplify the quantity of the parts.
3. Culture and selection. Microorganisms that have acquired the parts (in some cases there will be mistakes) grow in a medium and are selected.
4. Isolation of plasmid DNA. Microorganism's parts are extracted.
5. Comprobative enzymatic digestion. Parts are cut to separate.
6. Agarose gel electrophoresis. This technique allows to separate and visualize the different cut and separated parts to check that everything is correct.

GENETIC CONSTRUCTIONS

THE TWO STEPS

GENETIC CONSTRUCTION 1
Escherichia coli (derivative of DH5α) is the bacteria that acts as a chassis for ensemble the
genetic constructions. Intending to familiarise with it, we built a first test (Genetic construction 1)
To build a construction, digesting and joining the parts two by two is needed. In this construction, we digest and join Yeast Promoter TEF2 and RBS on one side, and Coding Sequence mCherrry and Yeast Terminator TEF1 on the other. Everything gets ensembled together in the end.

An antibiotic is poured into the culture medium which resistance has the plasmids with the parts that are introduced into the microorganism. Only those which acquired the plasmid will grow.

GENETIC CONSTRUCTION 2
Once we mastered the result from the first construction it is introduced in the yeast Yarrowia lipolytica. Its job will be getting fed from the lipids of waste cooking oil and synthesizes the beta-carotene.

SC-URA Uracile medium will allow Yarrowia lipolytica with genetic construction to grow. For the one without, we also chose YP(D) rich medium.

GENETIC
CONSTRUCTION 1

![](https://static.igem.org/mediawiki/2021/7/78/T--UMA_MALAGA--geneticcons1.png)

GENETIC
CONSTRUCTION 2

![](https://static.igem.org/mediawiki/2021/d/d0/T--UMA_MALAGA--geneticcons2.png)

WORKFLOW FOR BIOPAINT

THE LAST STEP

1. Extraction and isolation of beta-carotene from Yarrowia lipolytica.
2. Preparation of casein-based bio-paint.
3. Optimization of culture medium: Substitute glucose for WCO and evaluate the optimal
concentration for beta-carotene synthesis.

CONTACT US

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# Team:UMA MALAGA/Experiments

Wiki Team UMA

EXPERIMENTS

PROTOCOLS AND SAFETY

If future iGEM teams want to work with Yarrowia lipolytica and know protocols about basic techniques of molecular biology, this is their site!

[PDF](https://static.igem.org/mediawiki/2021/f/f1/T--UMA_MALAGA--protocol.pdf)

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# Team:UMA MALAGA/Results

Wiki Team UMA

RESULTS

OUR WORK

1. Backbone plasmid preparation

Bacteria transformation with backbone plasmids.

First of all, we transform Escherichia coli DH5α with three
different plasmids availables in iGEM plates: pSB1A10,
pSB1K3, and pSB1C3. Each plasmid has an antibiotic
resistance cassette, that is ampicillin, kanamycin, and
chloramphenicol resistance, respectively. The plasmid
features are collected in the table below.

![](https://static.igem.org/mediawiki/2021/0/0a/T--UMA_MALAGA--rt1.png)

Backbone plasmids miniprep

After plating with selective medium, and the growth of
bacterial colonies, we pick some colonies, and put overnight
cultures for plasmid extraction or miniprep. The DNA
concentration, A260/280 ratio, and A260/230 ratio of each
miniprep are collected in the table below.

![](https://static.igem.org/mediawiki/2021/5/59/T--UMA_MALAGA--rt2.png)

2. Test DNA construction

To ensure that the Ori and the URA marker we use are
compatible with the yeast strain Yarrowia lipolytica, we
have created a test construct to test the correct expression
within the yeast. This construction contains a promoter, a
RBS, the mcherry gene, and a terminator that we have to join
in an only part. The information about these parts are in
the table below.

![](https://static.igem.org/mediawiki/2021/0/08/T--UMA_MALAGA--rt3.png)

First of all, we transform Escherichia coli DH5α with these
different parts, we pick some colonies, and put overnight
cultures for plasmid extraction. The DNA concentration,
A260/280 ratio, and A260/230 ratio of each miniprep are
collected in the table below.

![](https://static.igem.org/mediawiki/2021/5/51/T--UMA_MALAGA--rt4.png)

When plasmids are isolated we run an electrophoresis
comprobative gel with three different isolations of each
plasmid in two different conditions, with and without a
digestion process with EcoRI and Pst1. To ensure the correct
state of these plasmids and the parts within them. The gels
of each part are below.

![](https://static.igem.org/mediawiki/2021/8/8f/T--UMA_MALAGA--ri1.png)

![](https://static.igem.org/mediawiki/2021/2/20/T--UMA_MALAGA--ri2.png)

The next step is to join these parts in one. For this aim,
we first combined parts two by two, promoter-RBS,
mcherry-terminator, and then these two parts in one. Two
parts cloning was carried out in pSB1A10 by digesting the
upstream part with EcoRI and Sptl1, the downstream part with
XbaI and Pst1, and the destination plasmid (pSB1A10) with
EcoRI and Pst1. A representative scheme of cloning
digestions is found below.

![](https://static.igem.org/mediawiki/2021/d/d4/T--UMA_MALAGA--ri3.png)

We transform Escherichia coli DH5α with these different
parts, we pick some colonies, and put overnight cultures for
plasmid extraction. The DNA concentration, A260/280 ratio,
and A260/230 ratio of each miniprep are collected in the
table below.

![](https://static.igem.org/mediawiki/2021/0/02/T--UMA_MALAGA--rt5.png)

When plasmids are isolated we run an electrophoresis
comprobative gel with three different isolations of each
plasmid in two different conditions, with and without a
digestion process with EcoRI and Pst1. To ensure the correct
state of these plasmids and the parts within them. The gels
of each part are below.

![](https://static.igem.org/mediawiki/2021/f/f4/T--UMA_MALAGA--ri4.png)

pTEF2-RBS PSB1A10 bands at 5.6 kb. The same band appears
in the digestion lanes, so it is proposed that the
digestion is partial, as well as a band at 0.4 kb that
corresponds to pTEF2 + RBS. Colony 2 is discarded
because it lacks it.

![](https://static.igem.org/mediawiki/2021/2/28/T--UMA_MALAGA--ri5.png)

mCherry-TEF1t PSB1A10 bands at 6.5 kb. Colony 1 is
discarded because it lacks it. At digestions lanes
mCherry and TEF1t and the backbone band at 1.2 kb and
5,3 kb, respectively.

In both gels, around 2 kb bands appear in the digestions
lanes. We believe that it is the previous plasmids, pSB1C3.

After that, we join these two parts in a final one, so we
combine the promoter-RBS part, and the mcherry-terminator
part in the destination plasmid pSB1K3 by digesting the
upstream part with EcoRI and Sptl1, the downstream part with
XbaI and Pst1, and the destination plasmid (pSB1K3) with
EcoRI and Pst1.

We transform Escherichia coli DH5α with this part, we pick
some colonies in kanamicin selective plates, and put
overnight cultures for plasmid extraction. The DNA
concentration, A260/280 ratio, and A260/230 ratio of each
miniprep are collected in the table below.

![](https://static.igem.org/mediawiki/2021/7/7d/T--UMA_MALAGA--rt6.png)

When plasmids are isolated we run an electrophoresis
comprobative gel with three different isolations of each
plasmid in two different conditions, with and without a
digestion process with EcoRI and Pst1. To ensure the correct
state of these plasmids and the parts within them. The gels
of each part are below.

![](https://static.igem.org/mediawiki/2021/a/a3/T--UMA_MALAGA--ri6.png)

pTEF2-RBS-mCherry-TEF1t pSB1K3 band at 3,8 kb.
pTEF2-RBS-mCherry-TEF1t part and the backbone pSB1KE by
separate band at 1,6 kb, and 2,2 kb, respectively. Colonies
1 and 2 are discarded due to no matching results. At
digested lanes, 1,6 kb bands don't exist, and instead of
that we have 1,1 kb bands. It is probably a digestion error.

FUTURE PLANS FOR THE PROJECT

The aim of lycopene and β-carotene pigments production in
Yarrowia lipolytica is carried out through the elaboration
of a construction with three genes encoding three enzymes
that catalyze the synthesis of both pigments from
geranylgeranyl-diphosphate (GGPP). These enzymes are
phytoene synthase, phytoene desaturase and lycopene cyclase.

Bacterial phytoene synthase is encoded by crtB gene and
catalyzes the first specific step in the carotenogenic
pathway by carrying out the condensation of two GGPP
molecules into one phytoene molecule. The phytoene´s
desaturation makes lycopene show up. Lycopene is the
previous molecule to β-carotene synthesis. In most organisms
that have the carotene synthesis pathway, the desaturation
is carried out by the joint action of some desaturase
enzymes. However, in bacteria this process is carried out by
a single phytoene synthase encoded by crtI gene. The last
enzyme that participates in the β-carotene synthesis is
lycopene cyclase encoded, in bacteria, by carRP gene. This
enzyme catalyzes the lycopene transformation into a
six-carbon ring.

To achieve this aim we will clone a Yarrowia lipolytica
vector into Escherichia coli. This plasmid has an URA3 gene
because the Yarrowia lipolytica strain we have has not de
capacity od URA3 production.After that we will generate a
construction with the genes implicated in beta carotene
production. These genes optimization is already done, as
well as the synthesis di novo trough IDT.

When the construction is ready, next step is simple, to
transform Yarrowia with that construction, extract the
pigments , and elaborate paint with these pigments.

CONTACT US

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