iGEM 2021 — Team MADRID_UCM

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:MADRID UCM/Notebook

[![](https://static.igem.org/mediawiki/2021/thumb/b/bd/T--MADRID_UCM--logo_simple_white.png/1200px-T--MADRID_UCM--logo_simple_white.png)](https://2021.igem.org/Team%3AMADRID_UCM)

* [Home](https://2021.igem.org/Team%3AMADRID_UCM)
* Project
  + [Description](https://2021.igem.org/Team%3AMADRID_UCM/Description)
  + [Metabollic Engineering](https://2021.igem.org/Team%3AMADRID_UCM/Design)
  + [Implementation](https://2021.igem.org/Team%3AMADRID_UCM/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AMADRID_UCM/Proof_Of_Concept)
* People
  + [Human Practices](https://2021.igem.org/Team%3AMADRID_UCM/Human_Practices)
  + [Attributions](https://2021.igem.org/Team%3AMADRID_UCM/Attributions)
  + [The 4C\_Fuels Team](https://2021.igem.org/Team%3AMADRID_UCM/Team)
  + [Collaborations](https://2021.igem.org/Team%3AMADRID_UCM/Collaborations)
  + [Divulgation & Education](https://2021.igem.org/Team%3AMADRID_UCM/Communication)
* WET Lab
  + [Genetic Design](https://2021.igem.org/Team%3AMADRID_UCM/SynbioDesign)
  + [Encapsulation](https://2021.igem.org/Team%3AMADRID_UCM/Encapsulation)
  + [Engineering](https://2021.igem.org/Team%3AMADRID_UCM/Engineering)
  + [Notebook](https://2021.igem.org/Team%3AMADRID_UCM/Notebook)
  + [Experiments](https://2021.igem.org/Team%3AMADRID_UCM/Experiments)
* DRY Lab
  + [Software](https://2021.igem.org/Team%3AMADRID_UCM/Software)
  + [Modelling](https://2021.igem.org/Team%3AMADRID_UCM/Model)
  + [Hardware](https://2021.igem.org/Team%3AMADRID_UCM/Hardware)
* iGEM
  + [Contribution](https://2021.igem.org/Team%3AMADRID_UCM/Contribution)
  + [Phototroph's SynBio](https://2021.igem.org/Team%3AMADRID_UCM/Plant)
  + [Sustainability](https://2021.igem.org/Team%3AMADRID_UCM/Sustainable)
  + [Parts](https://2021.igem.org/Team%3AMADRID_UCM/Parts)
  + [Results](https://2021.igem.org/Team%3AMADRID_UCM/Results)
  + [Safety](https://2021.igem.org/Team%3AMADRID_UCM/Safety)
  + [Awards](https://2021.igem.org/Team%3AMADRID_UCM/Awards)

Hardware - 4C\_FUELS

# 4C\_Fuels Notebooks

In this page you will find the laboratory notebooks, protocols and other information about how 4C\_Fuel project has been shaped from its initial idea to their final concept.

>

## Notebooks & Protocols

You can access to the laboratory notebooks of the project below:

[![DESCRIPCIÓN](https://static.igem.org/mediawiki/2021/d/d0/T--MADRID_UCM--image41.png)

##### Encapsulation Notebook](https://static.igem.org/mediawiki/2021/9/99/T--MADRID_UCM--Encapsulation_Notebook.pdf)

[![DESCRIPCIÓN](https://static.igem.org/mediawiki/2021/d/d0/T--MADRID_UCM--image41.png)

##### Synbio Notebook](https://static.igem.org/mediawiki/2021/f/f6/T--MADRID_UCM--SynBioLabNotebook.pdf)

During our laboratory work we have performed many different protocols. For most of us was our first time performing standard laboratory protocols from beginning to the end and because of that we have tested many different variations of eac procedure till optimizing its results.

 Most of performed protocols corresponds with already developed protocols by either other laboratory groups or iGEM teams. In the following links you could find those protocols which we consider that stands out of the conventional laboratory routine protocols.

[![DESCRIPCIÓN](https://static.igem.org/mediawiki/2019/0/06/T--MADRID_UCM--wp-content~uploads~2019~10~g6nS5Q8a_400x400.png)

##### Golden Gate Lv.0 Domestiction (Marburg)](https://www.protocols.io/view/golden-gate-lvl-0-8edhta6)

[![DESCRIPCIÓN](https://static.igem.org/mediawiki/2019/0/06/T--MADRID_UCM--wp-content~uploads~2019~10~g6nS5Q8a_400x400.png)

##### Golden Gate Lv.1/2 Domestiction (Marburg)](https://www.protocols.io/view/golden-gate-lvl-1-2-8d4hs8w)

[![DESCRIPCIÓN](https://static.igem.org/mediawiki/2021/f/f1/T--MADRID_UCM--PDF.svg)

##### Supercompentent *E. Coli* preparation](https://static.igem.org/mediawiki/2021/9/90/T--MADRID_UCM--adaptedInoueMethod.pdf)

[![DESCRIPCIÓN](https://static.igem.org/mediawiki/2019/0/06/T--MADRID_UCM--wp-content~uploads~2019~10~g6nS5Q8a_400x400.png)

##### Gibson Assembly (NEB)](https://www.protocols.io/view/Gibson-Assembly-Master-Mix-Assembly-E2611-imsupm)

[![DESCRIPCIÓN](https://static.igem.org/mediawiki/2019/0/06/T--MADRID_UCM--wp-content~uploads~2019~10~g6nS5Q8a_400x400.png)

##### Alginate-Sepiolite Beads Encapsulation](dx.doi.org/10.17504/protocols.io.bwqgpdtw)

[![DESCRIPCIÓN](https://static.igem.org/mediawiki/2019/0/06/T--MADRID_UCM--wp-content~uploads~2019~10~g6nS5Q8a_400x400.png)

##### Biocompatible Sepiolite-Chitosan Foams](dx.doi.org/10.17504/protocols.io.by5upy6w)

[![DESCRIPCIÓN](https://static.igem.org/mediawiki/2019/0/06/T--MADRID_UCM--wp-content~uploads~2019~10~g6nS5Q8a_400x400.png)

##### Cyanobacterial Yolk-Shell Preparation](https://www.protocols.io/view/cyanobacteria-yolk-shell-preparation-byr5pv86)

[![DESCRIPCIÓN](https://static.igem.org/mediawiki/2019/0/06/T--MADRID_UCM--wp-content~uploads~2019~10~g6nS5Q8a_400x400.png)

##### Cyanobacterial Encapsulation in Biocompatible Silica Gels](dx.doi.org/10.17504/protocols.io.by5vpy66)

We would like you to note that not all the utilized protocols have been extensively documented either in the lab notebooks or trhough *protocols.io*, however we have adapted and performed other procedures, optimizing them for our specific needs. IN this regard, we have adapted different protocols variants for *Polymerase Cycling Assembly (PCA)*, *Adapted 2-step Gibson Assembly for complex and GC rich fragments assembly*, *Touch-Down PCR protocols for amplification of PCC11801 genomic sequences* and an *alternative golden gate protocol*. If you are reading trhough our wiki and feel interested about any of this or others experimental procedures, please do not hesitate to contact us via: igemmadrid2021@gmail.com

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:MADRID UCM/Engineering

[![](https://static.igem.org/mediawiki/2021/thumb/b/bd/T--MADRID_UCM--logo_simple_white.png/1200px-T--MADRID_UCM--logo_simple_white.png)](https://2021.igem.org/Team%3AMADRID_UCM)

* [Home](https://2021.igem.org/Team%3AMADRID_UCM)
* Project
  + [Description](https://2021.igem.org/Team%3AMADRID_UCM/Description)
  + [Metabollic Engineering](https://2021.igem.org/Team%3AMADRID_UCM/Design)
  + [Implementation](https://2021.igem.org/Team%3AMADRID_UCM/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AMADRID_UCM/Proof_Of_Concept)
* People
  + [Human Practices](https://2021.igem.org/Team%3AMADRID_UCM/Human_Practices)
  + [Attributions](https://2021.igem.org/Team%3AMADRID_UCM/Attributions)
  + [The 4C\_Fuels Team](https://2021.igem.org/Team%3AMADRID_UCM/Team)
  + [Collaborations](https://2021.igem.org/Team%3AMADRID_UCM/Collaborations)
  + [Divulgation & Education](https://2021.igem.org/Team%3AMADRID_UCM/Communication)
* WET Lab
  + [Genetic Design](https://2021.igem.org/Team%3AMADRID_UCM/SynbioDesign)
  + [Encapsulation](https://2021.igem.org/Team%3AMADRID_UCM/Encapsulation)
  + [Engineering](https://2021.igem.org/Team%3AMADRID_UCM/Engineering)
  + [Notebook](https://2021.igem.org/Team%3AMADRID_UCM/Notebook)
  + [Experiments](https://2021.igem.org/Team%3AMADRID_UCM/Experiments)
* DRY Lab
  + [Software](https://2021.igem.org/Team%3AMADRID_UCM/Software)
  + [Modelling](https://2021.igem.org/Team%3AMADRID_UCM/Model)
  + [Hardware](https://2021.igem.org/Team%3AMADRID_UCM/Hardware)
* iGEM
  + [Contribution](https://2021.igem.org/Team%3AMADRID_UCM/Contribution)
  + [Phototroph's SynBio](https://2021.igem.org/Team%3AMADRID_UCM/Plant)
  + [Sustainability](https://2021.igem.org/Team%3AMADRID_UCM/Sustainable)
  + [Parts](https://2021.igem.org/Team%3AMADRID_UCM/Parts)
  + [Results](https://2021.igem.org/Team%3AMADRID_UCM/Results)
  + [Safety](https://2021.igem.org/Team%3AMADRID_UCM/Safety)
  + [Awards](https://2021.igem.org/Team%3AMADRID_UCM/Awards)

Engineering - 4C\_FUELS

![](https://static.igem.org/mediawiki/2021/9/9d/T--MADRID_UCM--gotita_bien_DEF_superdef.svg)

* Page Guide 👇🏼
* [1.I-SceI Unmarking System](#iscei)
* [2. The system in detail](#indetail)
* [3. DTBL Cycle](#dtbl)

# Engineering

During our project, we have developed genetic engineering strategies that might be useful for the synthetic biology community. In this page you will find how we have designed this gene edition tools and applied them to adress our own needs during labwork.

## I-SceI Unmarking System

##

### Industry demands markerless recombinant strains

Common laboratory practices handle genetically modified organisms that carry one or more antibiotic resistance. This is a common procedure to force mutant strains to maintain the foreing construction we transformed them with. Antibiotic resistance cassettes are left in the modified organisms. They could be seen as a marker left by the cloning and genetic modification procedures.

However, industrial demands are far from this traditional approach. When bioprocesses scale up, dependency on external substances like antibiotics or inducers turns into high operational costs. Moreover, if genetically modified organisms are going to be used for some specific purpose like feedstock or cosmetics they aren’t allowed to be cultured with antibiotics.

#### Current strategies

Because of the need to move on towards this antibiotic resistance-free scenario, some techniques and approaches have been developed to remove antibiotic resistance from mutant strains. One of the most popular for prokaryotic microorganisms is based on the introduction of the sacB gene from Bacillus subtilis, which codes for the levansucrase protein. sacB is constitutively expressed, and when bacterial cells are cultured in the presence of sucrose, sacB converts this sugar into a toxic compound. This acts as a counter-selection strategy. Once the mutants have been modified and selected using the antibiotic resistance, they are plated on sucrose to force the survival of only those colonies who have lost the antibiotic resistance-sacB cassette.

SacB unmarking strategy already works on prokaryotes such as cyanobacteria. We wanted to expand the repertoire of engineering tools available for phototrophs, focusing on unmarking strategies. That is why we looked for other systems that had not been tested on photosynthetic chassis. We found an interesting tool based on recombineering and I-SceI restriction enzyme. We aimed to adapt it to cyanobacteria, as well as improving it for the synthetic biology community.

##

### Our Unmarking system

Our aim is to adapt current I-SceI mediated recombination system for the generation of markerless mutants in cyanobacteria. Following our design could be possible to quickly obtain a markeless recombinants after two transformation and selection rounds. The key players of our system are a integration by single recombination and a rare restriction enzyme: I-SceI.

#### I-SceI: A 18bp rare cutting endonuclease

Unlike traditional restriction enzymes used in Molecular Biology, I-SceI is a rare cutting restriction enzyme that recognizes a 18 bp long sequence. This specific target has a chance to occur almost once in every 7\*10^10 base pairs. This fact makes I-SceI suitable for genetic engineering approaches that take place inside the cells. The chance of encountering its target sequence in our host’s genome is barely none. We can safely express it within our cells without disrupting the genome of the chassis.

I-SceI expression together with recombineering approaches can achieve high efficiency markerless genomic integrations or deletions. Here we present an overview of the procedure as well as our improvements and applications.

#### Single Recombination Integration

Genome integration techniques usually rely on double recombination events between synthetic constructs and the host’s genome. However, these events can be quite rare depending on the organism, and are definitely less common than single recombination events. I-SceI unmarking strategy relies on single recombination events.

If a plasmid carries an homology region and a single recombination event takes place, the whole plasmid gets inserted within the genome. **I-SceI cargo plasmids**  carry two I-SceI target sequences flanking the construct we want to integrate. This plasmid also carries an antibiotic resistance cassette, so recombinants could be selected.

#### How does the system work?

The proposed design is a 2-plasmid system, first an integrative plasmid for the selection of market mutants and secondly an I-SceI expression plasmid, which will drive the removal process of undesired marker genes. Eventually, this former plasmid must be curatated from the strain, a process which could become tedious. To overcome this limitation we have considered two simultaneous strategies to reduce procedure times and ease the selection of unmarked mutants.

A global perspective of the proposed system at work is depicted below

![ISceI overview](https://static.igem.org/mediawiki/2021/e/e0/T--MADRID_UCM--I-SceIOverview.png)

Briefly, the wild type is transformed by cosingle recombination with the integrative plasmid which harbors two homology arms and a a resistance cassete in the plasmid backbone. After that, transformants could be selected by resistance to this antibiotic. Then, the selected mutants would be transformed with the I-SceI replicative plasmid, and selected by the Chloramphenicol resistance of the I-SceI expression plasmid. Once transformants are obtained, I-SceI expression is induced in liquid culture. After a short time, I-SceI will generate double strand breaks in the DNA, forcing the DNA recombination, regenerating either the unmarked Wild-Type or mutant pehnotype. Eventually, I-SceI expression plasmid will be self-cured by the action of I-SceI on the I-SceI site present in expression plasmid backbone. Curated unmarked transformants could be then easily selected by plating in Agar-Sucrose. This is becaue the introduction of a SacB cassete within I-SceI expression plasmids impedes the non-curated cells to grow on sucrose.

##

### System Components

#### Adapted Acceptor Plasmids

![](https://static.igem.org/mediawiki/2021/d/dd/T--MADRID_UCM--I-SceIAcceptor.png)

Our aim is to make this system compatible with our assembly standard, then we have adapted the *Marburg Collection* Lv.1 and Lv.2 acceptor plasmids to serve as the single-recombination integrative vector. To do so, we have designed modified Lv. 1 and Lv. 2 Modular Cloning acceptor plasmids plasmids that harbor two I-SceI restriction sequences flanking the genetic device to integrate.

Since both of this plasmids are only required for co-integration, the standard Lv1 (AmpR/ColE1) / Lv2 (KanR/ColE1) *Marburg Collection* acceptor plasmids has been used as template for the introduction of I-SceI recognition sites.

#### I-SceI Expression Plasmid

![](https://static.igem.org/mediawiki/2021/1/1a/T--MADRID_UCM--I-SceIExpression.png)

In order to express I-SceI we have considered to adapt the system already build in pSEVA328S. This plasmid contains an I-SceI expression casete under the control of Pm/XylS system, which is induced by the pressence of benzoate and its derivates. Pm/XylS system has been reported to achieve fine regulation and show reduced leakiness in standard laboratory chassis. In addition, the plasmid harbors a Chloramphenicol resistance cassete (compatible with the MoClo Lv.1 and Lv.2 selection markers).

Our design contemplates to replace by cloning with AscI and FseI the origin of replication by the pANs Ori, documented in part [BBa\_K3228069](http://parts.igem.org/Part%3ABBa_K3228069). Likewise, a sacB cassete can be included by cloning with SanDI and SpeI in the *gadget position* of pSEVA328S

## The I-SceI Unmarking system in detail

An step by step description of how does our system behaves is shown below. The presented procedure corresponds with our theoretical design of I-SceI unmarking system, which could be virtually employed for almost any prokaryotic organism capable of homologous recombination.

##

### 1. Marked Mutants Generation

First step of the process is the transformation of the wild type strain with the integrative plasmid harboring the desired expression construct. During this transformation, the aim is to allow the cells to co-integrate the plasmid within their genetic material, acquiring the whole plasmid.
Because of this, selection can be then performed by plating antibiotic containing media.

![](https://static.igem.org/mediawiki/2021/f/fc/T--MADRID_UCM--I-SceIStep1.png)

##

### 2. I-SceI expression, DSB and recombination

![](https://static.igem.org/mediawiki/2021/e/e0/T--MADRID_UCM--I-SceIStep2.png)

Once recombinants are selected, we proceed by transforming them with **I-SceI expression plasmid**. This plasmid carries an I-SceI expression cassette under the control of a tightly regulated inducible promoter, and a second antibiotic resistance. By exposing cells to both antibiotics we can select those colonies that harbor both I-SceI expression plasmid and I-SceI cargo plasmid integrated in the genome.

The I-SceI cassette is then induced and the enzyme is expressed. I-SceI will cut the two target sequences that were flanking the construct to be integrated, causing Double Strand Breaks (DSBs) in the host’s genome.

![](https://static.igem.org/mediawiki/2021/0/02/T--MADRID_UCM--I-SceIStep3.png)

At this point the cell must repair the DSB in order to survive. Using the Homology-Directed Repair (HDR) machinery, cells can overcome this problem. Due to how the I-SceI cargo plasmid is designed, there are two ways cells can repair the DSB: one of them reverts the integration to a wild-type version, and the other leaves our desired construct markerlessly integrated in the genome. This process depends on chance and in theory it should render a 50% chance of success.

In our case I-SceI expression plasmid has been designed in a double origin of replication plasmid: pRO1600/ColE1 for replication in  *E. coli*  cloning strains, and pANS origin of replication. The latter has been demonstrated to be a functional origin of replication in *Synechococcus*. In this way we aim to express I-SceI enzyme within cyanobacteria.

##

### 3. I-SceI expression plasmid curation

One of the main problems of I-SceI systems is the strain curation process. After the counterselection I-SceI expression plasmid remains within the cell. Curation is usually done by passing cells several times without antibiotics to encourage them to lose the I-SceI expression plasmid. However, this relies on pure chance and can be quite a long process. For common heterotrophs like *E. coli*  or  *P. putida*  this can be achieved in a week, approximately. But when working with organisms like cyanobacteria the whole process could take much longer because of their long replication times.

This is why we decided to incorporate some novel features in our design such as a I-SceI target sequence within I-SceI expression plasmid to achieve self-curation. Also, we implemented a SacB expression cassette within I-SceI expression plasmid to force plasmid curation by plating mutants in the presence of sucrose at the end of the process.

However, the introduction of this features implies that I-SceI expression must be tightly controlled, since excessive expression or a leaky promoter baseline expression could drive the plasmid curation even before the double strand break event takes place.

## Design Build Test & Learn Cycle: Our Iteration

In our project we have embraced the Design Build Test and Learn (DTBL) principles for all of our laboratory work. Our aim is to iteratively develop our technology, starting with the construction of a purely theoretical design and refining the design according to the observed outcomes.

Particularly we have applied DTBL philosophy for the experimental design for the **validation of newly identified neutral sites** (read more about it in our [Software page](https://2021.igem.org/Team%3AMADRID_UCM/Software)) and the evaluation of I-SceI Unmarking System performance. In this way, our aim was to combine simultaneously both systems, co-integrating multiple plasmids harboring the identified neutral sites homology regions, as well as a control plasmid with the well defined Neutral Integration site I for *Synechococcus e. PCC11801*.

However, issued during cloning and delays in strain acces limited our possibility to perform a full iteration to DBTL cycle. On the one hand, digestion-ligation cloning of Lv.1/Lv.2 entry vectors didn't yielded any transformants harboring the desired I-SceI flanked dropout. On the other hand, we succesfully assembled the I-SceI expression plasmid with pANs/ColE1 origin of replication and an internal I-SceI recognition site. However the Pm/XylS system demonstrated to be leaky enough in different cloning *E. Coli* strains to compromise cell survival. This is because the baseline "leaky" expression of I-SceI was enough to self-destreoy the expression plasmid, which is ultimately responsible for cell survival during selection with chloramphenicol.

We observed how strains transformed with I-SceI expression plasmid harboring an internal I-SceI site did not grown properly on solid agar.

After designing and building the expression plasmid, we have observed how the streaks grow remarkably slow and they did not survive when inoculated in liquid media containing chloramphenicol. This results encouraged us to re-design the expresion plasmid in two different ways:

First, replacing the Pm/XylS system, screening for more tightly regulated promoters either in *E. Coli* and cyanobacteria. A reduced expression background is a requirement for succesful transformation with the plasmid.

Second, removing the internal I-SceI site of the expression plasmid, and relying solely on SacB cassete, which allows to select colonies by its sensitivity/insensitivity to sucrose. In this case, after I-SceI induction, bacteria should be cultured longer in liquid media without antibiotics, in order to ease the plasmid curation. Then, only those strains which have lost the plasmid will survive when plated.

References

Sun, T., Li, S., Song, X., Diao, J., Chen, L., Zhang, W., 2018. Toolboxes for cyanobacteria: Recent advances and future direction. Biotechnol. Adv. 36, 1293–1307. https://doi.org/10.1016/J.BIOTECHADV.2018.04.007

Jones, C.M., Parrish, S., Nielsen, D.R., 2021. Exploiting Polyploidy for Markerless and Plasmid-Free Genome Engineering in Cyanobacteria. ACS Synth. Biol. 10, 2371–2382. https://doi.org/10.1021/ACSSYNBIO.1C00269/SUPPL\_FILE/SB1C00269\_SI\_001.PDF

Chen, Z., Ling, W., Shang, G., 2016. Recombineering and I-SceI-mediated Pseudomonas putida KT2440 scarless gene deletion. FEMS Microbiol. Lett. 363. https://doi.org/10.1093/FEMSLE/FNW231

Gawin, A., Valla, S., Brautaset, T., 2017. The XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering. Microb. Biotechnol. 10, 702–718. https://doi.org/10.1111/1751-7915.12701

Egger, E., Tauer, C., Cserjan-Puschmann, M., Grabherr, R., Striedner, G., 2020. Fast and antibiotic free genome integration into Escherichia coli chromosome. Sci. Reports 2020 101 10, 1–10. https://doi.org/10.1038/s41598-020-73348-x

Lea-Smith, D.J., Vasudevan, R., Howe, C.J., 2016. Generation of Marked and Markerless Mutants in Model Cyanobacterial Species. JoVE (Journal Vis. Exp. 2016, e54001. https://doi.org/10.3791/54001

Dempwolff, F., Wischhusen, H.M., Specht, M., Graumann, P.L., 2012. The deletion of bacterial dynamin and flotillin genes results in pleiotrophic effects on cell division, cell growth and in cell shape maintenance. BMC Microbiol. 12, 298. https://doi.org/10.1186/1471-2180-12-298

Elnitski, L., Hardison, R.C., Li, J., Yang, S., Kolbe, D., Eswara, P., O’Connor, M.J., Schwartz, S., Miller, W., Chiaromonte, F., 2003. Distinguishing Regulatory DNA From Neutral Sites. Genome Res. 13, 64–72. https://doi.org/10.1101/GR.817703

Chaves, J.E., Wilton, R., Gao, Y., Munoz, N.M., Burnet, M.C., Schmitz, Z., Rowan, J., Burdick, L.H., Elmore, J., Guss, A., Close, D., Magnuson, J.K., Burnum-Johnson, K.E., Michener, J.K., 2020. Evaluation of chromosomal insertion loci in the Pseudomonas putida KT2440 genome for predictable biosystems design. Metab. Eng. Commun. 11, e00139. https://doi.org/10.1016/J.MEC.2020.E00139

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:MADRID UCM/Experiments

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* [Home](https://2021.igem.org/Team%3AMADRID_UCM)
* Project
  + [Description](https://2021.igem.org/Team%3AMADRID_UCM/Description)
  + [Metabollic Engineering](https://2021.igem.org/Team%3AMADRID_UCM/Design)
  + [Implementation](https://2021.igem.org/Team%3AMADRID_UCM/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3AMADRID_UCM/Proof_Of_Concept)
* People
  + [Human Practices](https://2021.igem.org/Team%3AMADRID_UCM/Human_Practices)
  + [Attributions](https://2021.igem.org/Team%3AMADRID_UCM/Attributions)
  + [The 4C\_Fuels Team](https://2021.igem.org/Team%3AMADRID_UCM/Team)
  + [Collaborations](https://2021.igem.org/Team%3AMADRID_UCM/Collaborations)
  + [Divulgation & Education](https://2021.igem.org/Team%3AMADRID_UCM/Communication)
* WET Lab
  + [Genetic Design](https://2021.igem.org/Team%3AMADRID_UCM/SynbioDesign)
  + [Encapsulation](https://2021.igem.org/Team%3AMADRID_UCM/Encapsulation)
  + [Engineering](https://2021.igem.org/Team%3AMADRID_UCM/Engineering)
  + [Notebook](https://2021.igem.org/Team%3AMADRID_UCM/Notebook)
  + [Experiments](https://2021.igem.org/Team%3AMADRID_UCM/Experiments)
* DRY Lab
  + [Software](https://2021.igem.org/Team%3AMADRID_UCM/Software)
  + [Modelling](https://2021.igem.org/Team%3AMADRID_UCM/Model)
  + [Hardware](https://2021.igem.org/Team%3AMADRID_UCM/Hardware)
* iGEM
  + [Contribution](https://2021.igem.org/Team%3AMADRID_UCM/Contribution)
  + [Phototroph's SynBio](https://2021.igem.org/Team%3AMADRID_UCM/Plant)
  + [Sustainability](https://2021.igem.org/Team%3AMADRID_UCM/Sustainable)
  + [Parts](https://2021.igem.org/Team%3AMADRID_UCM/Parts)
  + [Results](https://2021.igem.org/Team%3AMADRID_UCM/Results)
  + [Safety](https://2021.igem.org/Team%3AMADRID_UCM/Safety)
  + [Awards](https://2021.igem.org/Team%3AMADRID_UCM/Awards)

Experiments - 4C\_FUELS

![](https://static.igem.org/mediawiki/2021/9/9d/T--MADRID_UCM--gotita_bien_DEF_superdef.svg)

* Page Guide 👇🏼
* [1.Encapsulation Experiments](#encapsulation)
* [2. Metabolic Engineering Validation](#metmodel)
* [3. Genetic Tools Performance](#engsystems)

# Experiments

Experimental design is key when planning a scientific project like ours. Ideas are great, but there has to be a viable way to test them. If not, they will remain just like that: ideas. In this page you will find the experiments we have carried out, as well as others that we planned but haven't got the chance to test.

## Cell Encapsulation Experiments

An important ambit of 4C\_Fuels project is to settle the foundations for the development of photobiocatalytic materials. During this year we have succesfully designed synthetesis protocols for the creation of various materials with photobiocatalytic potential.

 In order to asses the potential of this materials for photobiocatalysis, we have evaluated its performance in terms of: Mechanical Properties, stability and cell viability, diffusional limitations and structural characterization.

##

### Viability Test: Assesing Mechanical & Biological Stability

In order to obtain conclusions about the viability of the cells inside all materials a simple experiment was designed. This experiment consisted of synthetizing fresh materials all at once and keeping them with mild agitation at 28 ºC, 50 uE of illumination during an entire month. Tubes were open for 30 seconds without sterile conditions once every 5 days, in order to allow gas exchange and expose the materials to normal environmental conditions.

 We have taken pictures of all the materials over the time in order to see how they are affected and how well they hold up. This way, we aim to either evaluate its mechanical and biological stability. The picture below shows the evolution of the different materials.

![Viabilitytest](https://static.igem.org/mediawiki/2021/0/0d/T--MADRID_UCM--materialviability.png)

![Yolk Viability](https://static.igem.org/mediawiki/2021/f/fd/T--MADRID_UCM--yolkviability.jpg)

Besides this results, we have also evaluated the yolk shell viability when kept in a buffer instead of growth media. We wanted to assess if the color retention of the material was just a sign of the stabilization of chlorophylls or an indication of encapsulated living cells. To do so, we aliquoted an equal amount of yolk-shell, centrifuged and resuspended it into BG-11 and also in PBS. They have been found to lose their colour in PBS in the priod of a week, while they retained their colour and continued producing gas bubbles, proof that the material can successfully keep cells alive for long periods of time.

The tube containing the BG-11 Yolk Shel suspension has been stored for more than 70 days without observing a decrease in Optical density or gas bubbling within the tube.

##

### Diffusion characterization

We need to know the diffusional limit of each material, in order to conclude which will be the ideal material to allow nutrients, CO2 and the synthesized product to travel through the materials. In short, we wanted to test the mass transfer limitations of the synthesized materials.

Then, it is necessary that when cells that secrete n-butanol are encapsulated, the material in which they are introduced must be sufficiently porous, and with an adequate pore size to be able to allow the diffusion of the generated product outside the material.

Since a butanol producing strain was not yet available, experiments involving dyes diffusion are therefore considered. Thus, materials have been directly synthesized with the dyes. During the experiment the material has been placed in culture media with mild agitation and dye’s diffusion process has been studied.

Aliquots of the supernatant are taken and subsequently analysed in a spectrophotometer to see the increase in dye concentration in the supernatant for each material analysed. These experiments have been developed based on Lambert Beer's law, which relates absorbance to concentration, using spectrophotometric methods.

##### Experimental protocol:

In the case of each experiment, a concentrated 10% dye solution has been prepared, from which 0.2 mL has been mixed with 3 mL of precursor of each material. Then materials has been synthesized following the developed protocols and fixed amount of 0.4g of each material has been used for the diffusion experiments.

##

#### The Results

After performing the experiments, the diffusional limitation of each material could be observed attending to the relative release of the colorant to the media. However it is important to notice that with these assay we are evalueating the overall transpor limitation of an specific morphological and physichochemical configuration for each inmovilization system.

 Materials like thin films will feature a higher surface-volume ratio, which eventually ease the release of the colorant to the media.

##### Graphic 1. BEADS with Congo red.

![diffusiongraphic](https://static.igem.org/mediawiki/2021/5/5b/T--MADRID_UCM--GRAPH_1.png)

As seen in the graphic, the amount of dye diffused into the medium is increasing as time increases. At some points in time the absorbance is lower compared to the linear trend. This may be due to various factors, such as the sampling or the decrease of volume in the medium each time it is sampled.

##### Graphic 2. BEADS with violet crystal

![diffusiongraphic](https://static.igem.org/mediawiki/2021/9/93/T--MADRID_UCM--GRAPH_2.png)

Unlike the previous one, the trend of these data is closer to a logarithmic exponential trend. There are also some points that don’t follow this, but the tendency is clear. It is important to note that the data with respect to graph 1 are similar. So, it can be concluded that the diffusional limit is almost the same with both dyes.

##### Graphic 3. THIN FILMS with violet crystal in water and BG11

Although some points are not at the trend, the increasing absorbance with time is also clear. The absorbance is eight times the absorbance shown on the beads, so, we can conclude that thin films have a lower diffusional limit.

It is remarkable that there exists a difference between the thin films in water and in BG-11. We suspect that this could be caused by hardening effects on the thin film material when exposed to BG-11. In addition the change in ionic force caued by BG-11 may also alter the migration of employed colorants.

![diffusiongraphic](https://static.igem.org/mediawiki/2021/2/2f/T--MADRID_UCM--GRAPH_3.png)

##### Graphic 4.SILICA GEL with Congo red

![diffusiongraphic](https://static.igem.org/mediawiki/2021/0/0b/T--MADRID_UCM--GRAPH_4.png)

This is the graph with the trend that most closely approximates linearity. There is a steady increase in the absorbance of the samples, and it is concluded that their diffusional limit is lower than that of the beads and is like the thin films.

Attending to the observed results, the highest diffusion rates (in terms of absolute absorbance) has been obtained by the *Thin Films* materials, followed by the silica gels.

##

### Morphological Characterization

In order to get an idea of the porosity and surface area of each material, as well as studying cell distribution and encapsulation success. The materials have been studied by SEM and optical microscopy.
In the SEM it has been possible to visualise what the surfaces look like, and to some extent, the porosity of each one. It can be seen whether the material features a lamellar structure, fibrous or compact. Likewise is observable how in some materials cells are easily visible, while in other cell viability is severely compromised.

 The following images corresponds with SEM and optical microscopy characterization of the synthesized materials.

![SEMImage](https://static.igem.org/mediawiki/2021/f/f4/T--MADRID_UCM--SEM-img1_beadnoFD.jpg)

##### Alginate Beads (No Freeze Dried)

![SEMImage](https://static.igem.org/mediawiki/2021/d/d8/T--MADRID_UCM--SEM-img1_beadsFD.jpg)

##### Alginate Beads (Freeze Dried)

![SEMImage](https://static.igem.org/mediawiki/2021/6/60/T--MADRID_UCM--SEM-img1_beadnocyano.jpg)

##### Alginate Beads Without Cyanobacteria (Freeze Dried)

![SEMImage](https://static.igem.org/mediawiki/2021/d/d8/T--MADRID_UCM--SEM-img1_beadsFD.jpg)

##### Thin Film Without Cyanobacteria

![SEMImage](https://static.igem.org/mediawiki/2021/3/34/T--MADRID_UCM--SEM-img1_thinfilnWITHcyano.jpg)

##### Thin Film with cyanobacteria

![SEMImage](https://static.igem.org/mediawiki/2021/d/d8/T--MADRID_UCM--SEM-img1_cyanofoam.jpg)

##### Chitosan-Sepiolite Foam with cyanobacteria

![SEMImage](https://static.igem.org/mediawiki/2021/5/54/T--MADRID_UCM--SEM-img1_gelcyano.jpg)

##### Silica Gel with Cyanobacteria (Frezze-Dried)

![SEMImage](https://static.igem.org/mediawiki/2021/f/f6/T--MADRID_UCM--SEM-img1_gelcyano_noFD.jpg)

##### Silica Gel with Cyanobacteria (No Frezze-Dried)

![SEMImage](https://static.igem.org/mediawiki/2021/7/7c/T--MADRID_UCM--SEM-img1_gelYS.jpg)

##### Silica Gel with Yolk-Shell (No Frezze-Dried)

![SEMImage](https://static.igem.org/mediawiki/2021/8/87/T--MADRID_UCM--SEM-img1_gelYSOptical.jpg)

##### Silica Gel with Yolk-Shell (No Frezze-Dried)

![SEMImage](https://static.igem.org/mediawiki/2021/2/28/T--MADRID_UCM--SEM-img1_YS.jpg)

##### Suspension of Yolk-Shell Microstructures

##

### Photobiocatalytic performance assays

Moving on from the previous characterization experiments, we decided to evaluate the performance of the photobiocatalysts in a real case scenario. To do so, we encapsulated a cyanobacteria strain able to secrete an easy-to-measure metabolite such as sucrose. We encapsulated exponentially growing cyanobacteria in Alginate-sepiolite beads, Yolk-Shell and Silica Gel Yolk-Shell materials, following the corresponding protocols. Biohybrid materials together with a non-encapsulated control were cultured in standard conditions.

During two weeks, supernatant samples were taken for each experimental sample. We recorded both OD720 and sucrose concentration, to then correlate sucrose production with biomass.

Our results showed that biohybrid materials did not compromise cyanobacteria growth. OD720 measures were taken for the course of two weeks for Yolk-Shells without reporting a decrease in growth. Their OD720 values remained constant at the initial value. For Alginate-sepiolite beads and Silica Gel Yolk-Shells OD measurements could not be taken due to the intrinsic material structure. However, they remained showing a vibrant green during the whole experiment.

Due to technical and timing issues, sucrose concentration could not be determined. Further research is needed to elucidate how each material affects metabolite secretion.

## Metabolic engineering Experiments

##

### n-Butanol Productivity Assays

We wanted to generate a butanol overproducing strain as a proof of concept for our phototrophic-based production platform. Sadly, due to cloning and strain transformation issues we ran out of time and no successful butanol-producing mutant cyanobacteria were obtained.

 However, we planned ahead the experiments needed for the characterization of butanol production. In the following section we are going to briefly summarize our experimental design to asses n-butanol productivity performance.

Butanol is not an easy molecule to measure: it has no color and gives no visible phenotype to cells. Also, we couldn’t find cheap methods or kits to measure this simple alcohol. Then, a suitable way to assess n-butanol production was culturing cells in closed flasks with bicarbonate supplementation at 25mM. This allows us to avoid n-butanol vaporization from the media. Using a culture volume of 50 mL,, every day each bottle will be opened and a 1.5 mL sample would be removed, replacing the extracted volume by fresh BG-11 - HEPES media supplemented with 250 mM bicarbonate.

The extracted sample would be centrifuged to remove biomass and supernatant would be extracted with dichloromethane (DCM) for 20 minutes of intermitente shaking. Then centrifugation will be used again to recover a clean DCM supernatant, which will be eventually spiked with isobutanol as internal standard for HPLC determination. In order to study the n-butanol concentration, an HPCL equipment using a ionic exclusion column with 4 % to 8 % cross-linked sulfonated styrene-divinylbenzene (SDVB), like *REZEX RFQ-Fast Acid H+ 8% column* and a Flame Ionization Detector could be used.

As discussed in [Metabolic Engineering](https://2021.igem.org/Team%3AMADRID_UCM/Design) we hypothesized that butanol biosynthesis could be improved by fine tuning pathways enzyme expression, by physically linking the two last enzymes of the biosynthesis pathway and by the introduction of synthetic bypasses to rewrite carbon utilization.

We designed multiple different constructs regarding these strategies. Our aim was to initially screen for the optimal performance mutant harboring a variant of n-butanol pathway alone. In this situation we aimed to compare the performance of the following strains.

* Pathway re-arranged Strains: Where each protein is preceded by its own RBS and thus gets translated individually. They correspond with strains harboring BOH3\_A, BOH3\_B, BOH2AB, BOH1ABC construct variants, explained within the [Genetic Design page](https://2021.igem.org/Team%3AMADRID_UCM/SynbioDesign).

* Linked constructs GSG: where the stop codon of PduP has been suppressed and the RBS from Slr1192 has been replaced by a short-flexible linker comprising three amino acids: GSG. Corresponding with the strains harboring BOH1ABC-GSG and BOH2AB-GSG

* Linked construct FR3F: similar as previous one but comprising a semi-rigid long linker: FRRRF (F-flexible GSG linker; R-rigid EAAAK linker). Corresponding with the strains harboring BOH1ABC-FR3F and BOH2AB-FR3F.

In the case of fusion enzynmes, we have previoysly carried out a bioinformatic analysis of the structure of the independent enzymes as well as both linker scenario strategies. No major conformational changes were found in the enzymes due to linker implementation.
To test these three strategies, we would just measure butanol production of each construct and compare it with the rest of the obtained mutant strains.

##

### Testing tolerance and secretion systems

Butanol is toxic to cells. One of its toxic effects is causing membrane instability, which affects phototrophic organisms to a greater extent due to their dependency on membranes (where the photosynthetic apparatus is located). That is the reason why we wanted to implement different strategies to improve the cell's tolerance to this alcohol.

Two proteins were chosen to be overexpressed in our modified cells: the chaperone HspA and the transcription factor SigB (see ‘Metabolic Engineering' to know more about them). Based on previous tolerance studies (refs.) we planned the following experiments to test whether their overexpression enhances butanol tolerance.

Cyanobacteria test strains and a wild-type control would be grown until 0.5 OD720. Then we would incubate our strains with butanol at different concentrations: 40 mg/L to resemble initial production concentrations, and 1.5 g/L as saturating concentration. Theoretically, after a 12h incubation period butanol has been able to diffuse and the correspondent butanol concentration must be present inside the cells. At this point we would compare growth by measuring OD720. Our hypothesis is that cyanobacteria that overexpress HspA, SigB, or both, would exhibit an improved tolerance and hence a higher growth compared with that of a wild type control.

These results haven’t been tested yet, but several studies have already seen this effect (refs.).

Another important tolerance strategy was the implementation of the butanol transporter AcrBv2 (see ‘Metabolic Engineering' to know more about it). Unlike HspA and SigB, this strategy doesn’t improve tolerance directly. AcrBv2 secretes butanol outside the cell, causing an indirect tolerance effect.

We would test AcrBv2 constructs with the same technique we would use for HspA and SigB, hypothesizing a similar trend of enhanced tolerance.

However, like other transporter proteins AcrBv2 is known to cause membrane stress if overexpressed without tight control. That is why we decided to include three differently regulated constructs when designing the AcrBv2 module, which we achieved by introducing different promoters upstream AcrBv2 gene (see ‘SynBio Design’ for further details):

* Constitutive regulation with a well characterized promoter like J23100 or J23119. We expect AcrBv2 to cause a detrimental effect on growth due to membrane stress.

* Inducible regulation with an inducible promoter like Pbad. In this case we would test different induction conditions in order to approximately get the optimal expression to obtain an improved tolerance but not an impaired growth.

* Autoregulation based on membrane stress, using Pgntk promoter. With this promoter we would expect to get a similar effect to the optimal condition found with the inducible promoter, in a similar way as has previously found in available literature.

AcrBv2 is not only expected to improve tolerance, but also to remove butanol from the cell. Butanol secretion is a key feature of our project: it pushes the equilibrium of the biosynthetic pathway towards butanol production. This way, selecting the optimal expression of AcrBv2 is a crucial aspect to improve overall strain performance.

Once we get the best performing AcrBv2 construct we would address its secretion properties. From the same tolerance experiment, we would pellet the cells after the incubation period, lysate them and obtain the organic-soluble fraction. Then we would measure its butanol concentration. We expect to find a lower butanol concentration inside the cells that express AcrBv2 than in those of a wild type, due to AcrBv2-driven butanol secretion.

All the proposed hypotheses are based on results found in previous literature.

## Evaluation of Gene Edition Systems

Besides designing a a metabolic engineering approach for cyanobacterial n-butanol production, we also found the need for efficient tools for cyanobacterial genome edition. Because of this, we decided to develop resources which could help us to speed up our strain engineering workflow and become an useful tool for the synthetic biology community.

In our team we have created NSFinder, a program that looks for putative neutral sites within a given genome (see [Software](https://2021.igem.org/Team%3AMADRID_UCM/Software) to know how this works). We planned an experimental strategy to test how NSFinder would perform in selecting novel neutral sites in cyanobacteria. Also, this system is thought to work with new recombineering techniques like the I-SceI-based counter-selection strategy we implemented (see Engineering for detailed information).

##

### Evaluation of Neutral Sites and I-SceI Unmarking System

Thanks to pJAY plasmids (see [Genetic Design](https://2021.igem.org/Team%3AMADRID_UCM/SynbioDesign)) we would assess each identified putative neutral site through YFP expression. However, with these experiments we would also check for the efficiency of I-SceI-mediated integration in phototrophic microorganisms, something that, to our knowledge, has not been verified to date (see [Engineering](https://2021.igem.org/Team%3AMADRID_UCM/Engineering) for further details). Taking these together, the following experimental design had two objectives:

* Address whether predicted neutral sites are suitable for genomic integration, and evaluate its relative expression levels.

* Evaluate the efficiency of our engineering design as a genome integration tool, compared with traditional approaches.

* Compare the behavior of marked and unmarked strains when using different neutral sites.

NSFinder found more than 100 putative neutral sites in PCC11801’s genome. Among all of these, we selected four for their characterization. Thus, we would construct one pJAY plasmid for each of them. These plasmids would be integrated in the genome selecting for single recombination events. This process would create antibiotic resistant mutants. Afterwards, I-SceI expression plasmid would be introduced.

Once I-SceI cuts in their target regions, there is a 50% chance of getting unmarked mutants or a reverted wild type, depending on the way the homologous recombination is performed for the double strand break repair. As cyanobacteria generally have multiple copies of their chromosome, PCR checking is needed along the way to confirm complete segregation of unmarked mutants.

In the end, we would have four pJAY strains, each of them harboring a YFP expression cassette in a different putative neutral site of the genome. By carrying out fluorescence measurements of each of the strains we would be able to characterize the potential of either neutral site as an expression location within PCC11801’s genome.

Also, an I-SceI expression plasmid would be built, in order to express the I-SceI restriction enzyme. Once modified acceptor plasmids are integrated in the genome, I-SceI would cut DNA at its recognition sequence causing double-strand breaks. Repairing mechanisms would then drive recombination , leading to antibiotic counterselection, leaving an antibiotic-free insertion in the microorganism’s genome.

By using I-SceI counterselection strategy, we rely on single recombination events that would introduce the whole pJAY plasmid into the genome. Single recombinants might express two antibiotic resistances: the insert resistance together with the backbone resistance. In this way we could select single recombinants by exposure to both antibiotics. Also, colonies should be expressing YFP.

References

D. Kaczmarzyk, J. Anfelt, A. Särnegrim and E. Hudson, "Overexpression of sigma factor SigB improves temperature and butanol tolerance of Synechocystis sp. PCC6803", 2021.

"Enhancing Tolerance to Short-Chain Alcohols by Engineering the Escherichia coli AcrB Efflux Pump to Secrete the Non-native Substrate n-Butanol", ACS Publications, 2021.

"Using Transcriptomics To Improve Butanol Tolerance of Synechocystis sp. Strain PCC 6803 | Applied and Environmental Microbiology", Applied and Environmental Microbiology, 2021.

S. Boyarskiy, S. Davis López, N. Kong and D. Tullman-Ercek, "Transcriptional feedback regulation of efflux protein expression for increased tolerance to and production of n-butanol", 2021.

N. Lee, W. Gielow and R. Wallace, "Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli.", 2021.

P. Till, J. Toepel, B. Bühler, R. Mach and A. Mach-Aigner, "Regulatory systems for gene expression control in cyanobacteria", 2021.

"A Library of Tunable, Portable, and Inducer-Free Promoters Derived from Cyanobacteria", ACS Publications, 2021.

H. Huang, D. Camsund, P. Lindblad and T. Heidorn, "Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology", 2021.

X. Liu, R. Miao, P. Lindberg and P. Lindblad, "Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO2in cyanobacteria", 2021.

Kumar, M., Saini, S., Gayen, K., 2014. Acetone-butanol-ethanol fermentation analysis using only high performance liquid chromatography. Anal. Methods 6, 774–781. https://doi.org/10.1039/C3AY41717D

Srivastava, V., Amanna, R., Rowden, S.J.L., Sengupta, S., Madhu, S., Howe, C.J., Wangikar, P.P., 2021. Adaptive laboratory evolution of the fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for improved solvent tolerance. J. Biosci. Bioeng. 131, 491–500. https://doi.org/10.1016/J.JBIOSC.2020.11.012

M. Flickinger, J. Schottel, D. Bond, A. Aksan and L. Scriven, "Painting and Printing Living Bacteria: Engineering Nanoporous Biocatalytic Coatings to Preserve Microbial Viability and Intensify Reactivity", Biotechnology Progress, vol. 23, no. 1, pp. 2-17, 2007. Available: 10.1021/bp060347r

J. Rooke, A. Léonard, C. Meunier and B. Su, "Designing Photobioreactors based on Living Cells Immobilized in Silica Gel for Carbon Dioxide Mitigation", ChemSusChem, vol. 4, no. 9, pp. 1249-1257, 2011. Available: 10.1002/cssc.201000442

S. Vasilieva, E. Lobakova, A. Lukyanov and A. Solovchenko, "Immobilized microalgae in biotechnology", Moscow University Biological Sciences Bulletin, vol. 71, no. 3, pp. 170-176, 2016. Available: 10.3103/s0096392516030135

E. Ruiz-Hitzky, M. Darder, A. Alcântara, B. Wicklein and P. Aranda, "Recent Advances on Fibrous Clay-Based Nanocomposites", Organic-Inorganic Hybrid Nanomaterials, pp. 39-86, 2014. Available: 10.1007/12\_2014\_283

M. Darder, P. Aranda and E. Ruiz-Hitzky, "Bionanocomposites: A New Concept of Ecological, Bioinspired, and Functional Hybrid Materials", Advanced Materials, vol. 19, no. 10, pp. 1309-1319, 2007. Available: 10.1002/adma.200602328

C. Meunier, J. Rooke, A. Léonard, H. Xie and B. Su, "Living hybrid materials capable of energy conversion and CO2 assimilation", Chemical Communications, vol. 46, no. 22, p. 3843, 2010. Available: 10.1039/c001799j

D. Killeen, M. Frydrych and B. Chen, "Porous poly(vinyl alcohol)/sepiolite bone scaffolds: Preparation, structure and mechanical properties", Materials Science and Engineering: C, vol. 32, no. 4, pp. 749-757, 2012. Available: 10.1016/j.msec.2012.01.019

P. Johnson et al., "Spray-Dried Multiscale Nano-biocomposites Containing Living Cells", ACS Nano, vol. 9, no. 7, pp. 6961-6977, 2015. Available: 10.1021/acsnano.5b01139

M. Perullini, M. Calcabrini, M. Jobbágy and S. Bilmes, "Alginate/porous silica matrices for the encapsulation of living organisms: tunable properties for biosensors, modular bioreactors, and bioremediation devices", Open Material Sciences, vol. 2, no. 1, 2015. Available: 10.1515/mesbi-2015-0003

J. Rooke, A. Léonard, H. Sarmento, C. Meunier, J. Descy and B. Su, "Novel photosynthetic CO2bioconvertor based on green algae entrapped in low-sodium silica gels", J. Mater. Chem., vol. 21, no. 4, pp. 951-959, 2011. Available: 10.1039/c0jm02712j

D. Dickson and R. Ely, "Silica sol-gel encapsulation of cyanobacteria: lessons for academic and applied research", Applied Microbiology and Biotechnology, vol. 97, no. 5, pp. 1809-1819, 2013. Available: 10.1007/s00253-012-4686-8

M. Darder et al., "Algae–silica systems as functional hybrid materials", J. Mater. Chem., vol. 20, no. 42, pp. 9362-9369, 2010. Available: 10.1039/b913269d

L. Wang et al., "Single-cell yolk-shell nanoencapsulation for long-term viability with size-dependent permeability and molecular recognition", National Science Review, vol. 8, no. 4, 2020. Available: 10.1093/nsr/nwaa097

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# Team:MADRID UCM/Results

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Cloning design - 4C\_FUELS

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* Page Guide 👇🏼
* [1. Social Results](#hpresults)
* [2. Wet Lab Results](#wetlab)
* [3. Dry Lab Results](#drylab)

# Results

iGEM is not an easy journey and often all the planification does not ends in the expected results, but that's how science work and every unexpected result is actually an opportunity to understand further and discover something new.

In 4C\_Fuels we have found multiple difficulties in our road towards our final goal. However after an entire year working in the project we have made important achievements, which we aim they will inspire and ease the work of future iGEM teams. In this page you will find the most relevant outcomes of 4C\_Fuels project.

## 4C\_Fuels Project: The Results

After thoughtfully designing all the project contents and workplan, our team started working in all of its aspects. The results of all this work are furtherly detailed in the [Results](https://2021.igem.org/Team%3AMADRID_UCM/Results)  page, as well as in ever page of this wiki. However, a brief summary is explained below

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### HUMAN PRACTICE & SOCIAL ASPECTS

As we have deeply explained within our [sustainability page](https://2021.igem.org/Team%3AMADRID_UCM/Sustainable)  . 4C\_Fuels was born to achieve a more sustaibable society, and this could only be achieved considering not only the technoeconomic aspect of sustainability, but also the social one.

 With this idea in mind, we have developed various contents with the aim to promote social awareness about sustainability and raise the interest of general society for the potential of science and technology for solving our most pressing challenges. However, we also aimes to transmit the idea that technology alone is useless, unless people is aware about how its opinion, behavior and ideas are crucial for the definition of how technology is developed, and how it can be applied to transform our world.

In addition, we truly believe in the potential of iGEM to fuel this sustainable transition our world needs. Because of this we have taken an active role within iGEM community (know more about in the [Collaborations page](https://2021.igem.org/Team%3AMADRID_UCM/Collaborations)  ), as well as promoted the iGEM concept within our region. Besides huge collaborative projects, like the participation in the **iGEM PHototrophs Community**, we have also offered various lectures in our region and created divulgative contents explaining what iGEM is. Our aim with all of these activities is not only to settle the foundations for the emergence of future iGEM teams in our region, but also to raise interest about synthetic biology in the society. We want people to understand how synthetic biology is a powerful tool that goe beyond the GMO debate and can actually offer solutions for many problems nowadays still unsolved.

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### WET LAB RESULTS

After trial and error a few times without clear and promising results, it is easy to give up but we did try until the end. The work at the lab has been conducted iteratively, solving small problems till reaching the next one. Even though the results expected were not a reality yet, we have validated part of our theorethical design and documented all the issues we have found. We believe that our theorethical design can still be validated by future iGEM teams, which will take all of our work one step further to reality. Because of this, all the theorethical design has been extensively explained in this wiki, to ease the work of future scientist in the development of sustainable biomanufacturing technologies.

Nevertheless all our wet lab work has yileded some interesting outcomes.

* **Cell Encapsulation.**On the one hand we have managed to study multiple procedures for [cyanobacteria encapsulation](https://2021.igem.org/Team%3AMADRID_UCM/Encapsulation)  in biohybrid materials. Our aim was the intial screening for the future development of cell-encapsulated photobiocatalyst. As a result of this research we have identified a promising silica-based nanomaterial where cyanobacterial cells can be kept alive for more than 30 days without losing its metabolic ability. All of this work has been extensively document in the [encapsulation page](https://2021.igem.org/Team%3AMADRID_UCM/Encapsulation)  .

* **Cyanobacteria Synthetic Biology** We believe in the need to expand the available toolbox for cyanobacteria synthetic biology applications. In this aim we have proposed the theoretical design of an efficient Unmarking System for cyanobacteria, as well as created extensive documentation about cyanobacterial metabolic engineering. In addition, we have also documented our experience accessing novel fast-growin strains, which may pose important advantages to speed up the development of cyanobacteria metabolic engineering field. All of this contents can be found within our contribution to the [Phototrophs Handbook](https://static.igem.org/mediawiki/2021/e/ef/T--MADRID_UCM--Phototrophs_Handbook_V1.0.pdf)  as well as in the[Phototrophs Synthetic Biology](https://2021.igem.org/Team%3AMADRID_UCM/Plant)  and [Engineering](https://2021.igem.org/Team%3AMADRID_UCM/Engineering)  pages.

* **Part Collection and Cloning design** Despite we have been unable to succesfully test our expression constructs within a cyanobacteria. We managed to succesfully assemble and sequence-verify a set of useful regulatory and coding parts, which are furtherly detailed within the [Contribution Page](https://2021.igem.org/Team%3AMADRID_UCM/Contribution)  . In addition, because of our specific need to express a large number of enzymes we have adapted specific cloning procedures which are compatible with the utilization of the [*Marburg Collection MoClo Toolbox*](https://2018.igem.org/Team%3AMarburg/Design)  . All of this strategies are furtherly explained at the [Cloning Design page](https://2021.igem.org/Team%3AMADRID_UCM/SynbioDesign)

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### DRY LAB RESULTS

Besides woring in the wet lab, we have also employed all tools in our hands to adress the needs of our project. In this regard, we have worked in three main ambits.

* **Hardware** Inspired by the work of previous iGEM teams we have commited ourselves to the task of optimizing and adapting for our needs a previous open-source photobioreactor system. In this regard, we have worked in the construction of a device which will help us to cultivate our cyanobacteria in tightly controlled conditions, as well as automatically record the relevant growth parameters. All this hardware work lead to the succesful construction of an improved version of the [OpenPBR system designed by Humboldt\_Berlin 2019 iGEM team](https://2019.igem.org/Team%3AHumboldt_Berlin/Hardware)  . To know more about our findings and improvements you can visit our [Hardware page](https://2021.igem.org/Team%3AMADRID_UCM/Hardware)

* **Software** Bioinformatics is gaining more and more importance for the development of any kind of work within synthetic and molecular biology. In fact we found ourselves in the need of identifying new integration sites for the novel fast-growing cyanobacterial strain we were planning to transform. To achieve this task we have developed a python script: **NSfinder**. This software piece is capable of identifying putative neutral integration sites in any prokaryotik genome, requiring only genomic sequence information. Our aim with the development of this tool, was to either fulfill one of our project needs as well as to provide synthetic biology community with an additional resource for easing the utilization of non-conventional strains during their research. To now more about this you can visit our [Software page](https://2021.igem.org/Team%3AMADRID_UCM/Software)

* **Modelling** Eventually we have also used computational tools in order to model and simulate the behavior of our design. First, we have colaborated with [MiamiU\_OH 2019 iGEM Team](https://2021.igem.org/Team%3AMiamiU_OH)  to model our metabolic design and study how our synthetic pathways will perform in the cyanobacteria.

   Secondly, we have deveoped an intiial engineering design to model and simulate a complete photosynthetic n-butanol production bioprocess, considering current performance of n-butanol producing engineered cyanobacteria. The results of this simulation validated the techno-economic viability of our proposed technology. Further information about all of this results can be found at the [Modelling page](https://2021.igem.org/Team%3AMADRID_UCM/Model)  and [Proof Of Concept Page](https://2021.igem.org/Team%3AMADRID_UCM/Proof_Of_Concept)  .