iGEM 2021 — Team Linkoping_Sweden

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# Team:Linkoping Sweden/Notebook

Team:Linkoping Sweden - 2021.igem.org

[![](https://static.igem.org/mediawiki/2021/thumb/2/2e/T--Linkoping_Sweden--project_logo_white.png/120px-T--Linkoping_Sweden--project_logo_white.png)](https://2021.igem.org/Team%3ALinkoping_Sweden)

* PROJECT
  + [Description](https://2021.igem.org/Team%3ALinkoping_Sweden/Description)
  + [Design](https://2021.igem.org/Team%3ALinkoping_Sweden/Design)
  + [Model](https://2021.igem.org/Team%3ALinkoping_Sweden/Model)
  + [Implementation](https://2021.igem.org/Team%3ALinkoping_Sweden/Implementation)
  + [Sustainabilty](https://2021.igem.org/Team%3ALinkoping_Sweden/Sustainable)
* LAB
  + [Results](https://2021.igem.org/Team%3ALinkoping_Sweden/Results)
  + [Improvement](https://2021.igem.org/Team%3ALinkoping_Sweden/Improvement)
  + [Contribution](https://2021.igem.org/Team%3ALinkoping_Sweden/Contribution)
  + [Engineering](https://2021.igem.org/Team%3ALinkoping_Sweden/Engineering)
  + [Notebook](https://2021.igem.org/Team%3ALinkoping_Sweden/Notebook)
  + [Protocols](https://2021.igem.org/Team%3ALinkoping_Sweden/Protocols)
  + [Safety](https://2021.igem.org/Team%3ALinkoping_Sweden/Safety)
* PARTS
  + [Parts Overview](https://2021.igem.org/Team%3ALinkoping_Sweden/Parts)
  + [Part Collection](https://2021.igem.org/Team%3ALinkoping_Sweden/Part_Collection)
  + [Basic Parts](https://2021.igem.org/Team%3ALinkoping_Sweden/Partsbasic)
  + [Composite Parts](https://2021.igem.org/Team%3ALinkoping_Sweden/Partscomposite)
  + [Vectors and Primers](https://2021.igem.org/Team%3ALinkoping_Sweden/vectorsprimers)
* PEOPLE
  + [Team Members](https://2021.igem.org/Team%3ALinkoping_Sweden/Team)
  + [Attributions](https://2021.igem.org/Team%3ALinkoping_Sweden/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3ALinkoping_Sweden/Collaborations)
  + [Sponsors](https://2021.igem.org/Template%3ALinkoping_Sweden/Spons)
  + [Team Album](https://2021.igem.org/Team%3ALinkoping_Sweden/Album)
* HUMAN PRACTICES
  + [Integrated Human Practices](https://2021.igem.org/Team%3ALinkoping_Sweden/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3ALinkoping_Sweden/Communication)
  + [Memory](https://2021.igem.org/Template%3ALinkoping_Sweden/Memory)
  + [Education](https://2021.igem.org/Team%3ALinkoping_Sweden/Education)
  + [Phototroph Handbook](https://2021.igem.org/Team%3ALinkoping_Sweden/Handbook)
* AWARDS
  + [Medal Criteria](https://2021.igem.org/Team%3ALinkoping_Sweden/Medal%20Criteria)

# Notebook

![](https://static.igem.org/mediawiki/2021/e/e2/T--Linkoping_Sweden--week10-13.png)

## Week 10-13

08.03 - 04.04

Discussed possible project ideas. Had brainstorming sessions, did a lot of research and finally decided to work with desalination of seawater using cyanobacteria mainly inspired by the [Shanghai 2015](https://2015.igem.org/Team%3ASJTU-BioX-Shanghai) team and an article published in Plant Physiology by J. Amezaga et al. titled *Biodesalination: a case study for applications of photosynthetic bacteria in water treatment.*

![](https://static.igem.org/mediawiki/2021/1/18/T--Linkoping_Sweden--notebook_idea.png)

## Week 15-18

12.04 - 09.05

More in-depth research regarding our project and SWOT analysis. Decided on more aspects of the project such as possible usage of fast-growing bacteria to validate the parts and ways of removing the bacteria from the desalinated water.

![](https://static.igem.org/mediawiki/2021/f/f2/T--Linkoping_Sweden--week19-21.png)

## Week 19-21

10.05 - 30.05

Had a tutorial on how to use Benchling and construct BioBricks. Started preparing protocols for the lab work and researched possible ways to improve an existing BioBrick. The logo of the project was finished.

![](https://static.igem.org/mediawiki/2021/0/06/T--Linkoping_Sweden--week22-24.png)

## Week 22-24

31.05 - 20.06

Lab work preparation. Continued designing BioBricks and working on protocols. Had the first meetings with experts in the field. The first week of the lab was mostly focused on learning the methods and preparing reagents. Decided to take part in the postcard project organised by iGEM Düsseldorf.

![](https://static.igem.org/mediawiki/2021/b/bb/T--Linkoping_Sweden--week25.png)

## Week 25

21.06 - 27.06

Received the cyanobacteria *Synechocystis sp. PCC 6803* and prepared knock-in plasmids pEERM1 and pEERM4. Released the first episode of the podcast. Decided on a plan for the mathematical modelling part of the project.

![](https://static.igem.org/mediawiki/2021/c/c6/T--Linkoping_Sweden--week26.png)

## Week 26

28.06 - 04.07

Prepared BG-11 media for *Synechocystis sp. PCC 6803*, restreaked them and prepared large-scale liquid cultures. Prepared our two other bacteria, *E. coli* and *V. natriegens*, for future transformations (competent cells). Continued contacting experts that could help in the improvement of different parts of the project and worked on our Integrated Human Practices.

![](https://static.igem.org/mediawiki/2021/6/6f/T--Linkoping_Sweden--week27.png)

## Week 27

05.07 - 11.07

Restreaked more wild type *Synechocystis sp. PCC 6803* as well as the S-layer knockout from *Synechocystis sp. PCC 6803*. Transformation of our improved part started and several BioBricks studied for our contribution, chromoproteins, were transformed into *E. coli*. Liquid cultures of *V. natriegens* and *E. coli* were made with and without a beta-carotene 15,15'-dioxygenase-containing plasmid for control purposes. Started planning the Nordic iGEM Conference together with the Chalmers-Gothenburg team.

![](https://static.igem.org/mediawiki/2021/7/7d/T--Linkoping_Sweden--week28.png)

## Week 28

12.07 - 18.07

Visited Gotland to learn more about the water shortage on the island and talked to multiple potential users, farmers. Also collected seawater samples for our experiments. After returning, performed mutagenesis to correct mutations in eforRed improved DNA construct and then transformed it into *E. coli* XL1 competent cells. Started testing the transformation protocol on *Synechocystis sp. PCC 6803* with pEERM1 and pEERM4 plasmids. Began our first experiments on ideal growth conditions of *Synechocystis sp. PCC 6803*.

![](https://static.igem.org/mediawiki/2021/b/be/T--Linkoping_Sweden--week29.png)

## Week 29

19.07 - 25.07

Ran an agarose gel on the eforRed improved DNA construct to confirm that the mutagenesis was successful. Purified the pEERM1 and pEERM4 plasmids that were amplified in XL1. Continued experimenting with the *Synechocystis sp. PCC 6803* transformation. Set up more growth experiments with different seawater samples and different conditions (different pH, salinity levels etc). Continued working on developing the modelling part of the project and on the podcast.

![](https://static.igem.org/mediawiki/2021/0/08/T--Linkoping_Sweden--week30.png)

## Week 30

26.07 - 01.08

Transformed CBD-containing plasmids into XL1 for amplification several times due to unsuccessful attempts. Amplified linear constructs from TwistBioscience with PCR. Prepared plasmids with CBD, eforRed improved (both correct and mutated versions – to confirm that mutagenesis was successful). Sent several BioBricks from the distribution kit (that we had transformed) for sequencing. Had a meeting with SUNY Oneonta and TU Darmstadt teams. Hosted the Nordic iGEM Conference together with the Chalmers-Gothenburg iGEM team.

![](https://static.igem.org/mediawiki/2021/8/80/T--Linkoping_Sweden--week31.png)

## Week 31

02.08 - 08.08

Continued working on CBD-containing plasmids with mutations because the previous mutagenesis was unsuccessful. Decided to order linear DNA fragments instead of performing mutagenesis to save time. Cloned MscL fragments into pEERM1 and pEERM4 plasmids, transformed into XL1 and verified the size of the obtained plasmids by gel electrophoresis. Transformed halorhodopsin pUCIDT plasmids into XL1. Started an experiment to monitor the growth of Synechocystis sp. PCC 6803 in autoclaved and non-autoclaved brackish and seawater using BG-11 1X as a control to obtain data for the modelling group. Continued with the transformation attempts in *Synechocystis sp. PCC 6803*.

![](https://static.igem.org/mediawiki/2021/d/d7/T--Linkoping_Sweden--week32.png)

## Week 32

09.08 - 15.08

Did colony screening of MscL in pEERM1 and pEERM4, beta-carotene in pEERM1 and pEERM4, and linker-GFP in pEERM1 and pEERM4 in XL1. Acquired data from chromoprotein characterization. Attempted to ligate linear CBD fragments into pEERM1 and pEERM4 and transform them into XL1. Continued with daily growth measurements of *Synechocystis sp. PCC 6803* in multiple types of seawater and BG-11 media. Acknowledged that the bacteria in non-autoclaved water did not survive long. Took microscopic images of seawater samples with no added *Synechocystis sp. PCC 6803* to find out what was killing the strain during the growth experiment. Established that the transformation attempts in *Synechocystis sp. PCC 6803* appeared to be unsuccessful.

![](https://static.igem.org/mediawiki/2021/a/ac/T--Linkoping_Sweden--week33.png)

## Week 33

16.08 - 22.08

Continued working on the colony screens that were started last week, and then plasmid prepped and made glycerol stocks of the colony screening products. Attempted to transform pEERM1 and pEERM4 into *Synechocystis sp. PCC 6803* following an alternate protocol. Ligated several parts with linker-GFP, like MscL in pEERM4. Continued with daily growth measurements of *Synechocystis sp. PCC 6803* in multiple types of seawater and BG-11 media. Held a presentation during a Phototroph Meetup - meetings between various iGEM teams working with phototrophs.

![](https://static.igem.org/mediawiki/2021/a/a1/T--Linkoping_Sweden--week34.png)

## Week 34

23.08 - 29.08

Decided to treat cleaved plasmids with calf intestinal phosphatase before ligation in order to avoid re-ligation of the plasmid without the insert. Switched to using pSB1C3 and pUC19 as cloning vectors after discovering a design flaw using pEERM1 and pEERM4. Transformed halorhodopsin in pUCIDT plasmid and MscL-Linker-GFP in pEERM4 into *V. natriegens*. Continued with daily growth measurements of *Synechocystis sp. PCC 6803* in multiple types of seawater and BG-11 media. Full-time lab work ended due to everyone going back to their studies.

![](https://static.igem.org/mediawiki/2021/f/f7/T--Linkoping_Sweden--week35.png)

## Week 35

30.08 - 05.09

Started the transformation of MscL in pEERM1 and pEERM4 into *Synechocystis sp. PCC 6803* using the alternate protocol and continued working on cleaving and ligating our linear DNA fragments into pUC19. Continued with daily growth measurements of *Synechocystis sp. PCC 6803* in multiple types of seawater and BG-11 media.

![](https://static.igem.org/mediawiki/2021/f/f2/T--Linkoping_Sweden--week36.png)

## Week 36

06.09 - 12.09

Ended with daily growth measurements of *Synechocystis sp. PCC 6803* in multiple types of seawater and BG-11 media. Visited high schools as part of our Education and Communication.

![](https://static.igem.org/mediawiki/2021/c/ca/T--Linkoping_Sweden--week37.png)

## Week 37

13.09 - 19.09

Continued working with our linear DNA fragments, attempting to cleave and ligate them into pUC19 plasmids. Attempted to transform our constructs into *E. coli*.

![](https://static.igem.org/mediawiki/2021/4/4d/T--Linkoping_Sweden--week38.png)

## Week 38

20.09 - 26.09

Sent plasmids for sequencing to check if they contained the correct inserts. The transformation of MscL in pEERM1 and pEERM4 into *Synechocystis sp. PCC 6803* turned out to be unsuccessful.

![](https://static.igem.org/mediawiki/2021/d/d7/T--Linkoping_Sweden--week32.png)

## Week 39

27.09 - 03.10

Continued working with our linear DNA fragments, attempting to cleave and ligate them into pUC19 plasmids. Attempted to transform the cloned constructs into *E. coli.* and *V. natriegens*. Studied the seawater samples with *Synechocystis sp. PCC 6803* from previous growth experiments under the microscope.

![](https://static.igem.org/mediawiki/2021/1/13/T--Linkoping_Sweden--week40.png)

## Week 40

04.10 - 10.10

Focused on perfecting and characterizing our improved part. Continued working on developing the wiki page and the presentation video.

![](https://static.igem.org/mediawiki/2021/f/fa/T--Linkoping_Sweden--Transparent_iGEM_Logo.png)

Life depends on WATER but the reservoir depends on YOU.

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# Team:Linkoping Sweden/Engineering

Team:Linkoping Sweden - 2021.igem.org

[![](https://static.igem.org/mediawiki/2021/thumb/2/2e/T--Linkoping_Sweden--project_logo_white.png/120px-T--Linkoping_Sweden--project_logo_white.png)](https://2021.igem.org/Team%3ALinkoping_Sweden)

* PROJECT
  + [Description](https://2021.igem.org/Team%3ALinkoping_Sweden/Description)
  + [Design](https://2021.igem.org/Team%3ALinkoping_Sweden/Design)
  + [Model](https://2021.igem.org/Team%3ALinkoping_Sweden/Model)
  + [Implementation](https://2021.igem.org/Team%3ALinkoping_Sweden/Implementation)
  + [Sustainabilty](https://2021.igem.org/Team%3ALinkoping_Sweden/Sustainable)
* LAB
  + [Results](https://2021.igem.org/Team%3ALinkoping_Sweden/Results)
  + [Improvement](https://2021.igem.org/Team%3ALinkoping_Sweden/Improvement)
  + [Contribution](https://2021.igem.org/Team%3ALinkoping_Sweden/Contribution)
  + [Engineering](https://2021.igem.org/Team%3ALinkoping_Sweden/Engineering)
  + [Notebook](https://2021.igem.org/Team%3ALinkoping_Sweden/Notebook)
  + [Protocols](https://2021.igem.org/Team%3ALinkoping_Sweden/Protocols)
  + [Safety](https://2021.igem.org/Team%3ALinkoping_Sweden/Safety)
* PARTS
  + [Parts Overview](https://2021.igem.org/Team%3ALinkoping_Sweden/Parts)
  + [Part Collection](https://2021.igem.org/Team%3ALinkoping_Sweden/Part_Collection)
  + [Basic Parts](https://2021.igem.org/Team%3ALinkoping_Sweden/Partsbasic)
  + [Composite Parts](https://2021.igem.org/Team%3ALinkoping_Sweden/Partscomposite)
  + [Vectors and Primers](https://2021.igem.org/Team%3ALinkoping_Sweden/vectorsprimers)
* PEOPLE
  + [Team Members](https://2021.igem.org/Team%3ALinkoping_Sweden/Team)
  + [Attributions](https://2021.igem.org/Team%3ALinkoping_Sweden/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3ALinkoping_Sweden/Collaborations)
  + [Sponsors](https://2021.igem.org/Template%3ALinkoping_Sweden/Spons)
  + [Team Album](https://2021.igem.org/Team%3ALinkoping_Sweden/Album)
* HUMAN PRACTICES
  + [Integrated Human Practices](https://2021.igem.org/Team%3ALinkoping_Sweden/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3ALinkoping_Sweden/Communication)
  + [Memory](https://2021.igem.org/Template%3ALinkoping_Sweden/Memory)
  + [Education](https://2021.igem.org/Team%3ALinkoping_Sweden/Education)
  + [Phototroph Handbook](https://2021.igem.org/Team%3ALinkoping_Sweden/Handbook)
* AWARDS
  + [Medal Criteria](https://2021.igem.org/Team%3ALinkoping_Sweden/Medal%20Criteria)

[Growth](#tab1)

[Toxicity](#tab2)

[Constructs](#tab3)

[References](#tab4)

## Engineering Success

During our project, we came across multiple difficulties that forced us to rethink our plan and the way we do things.
This is a natural part in any experimental research and it can be viewed as a part of the engineering cycle.

![](https://static.igem.org/mediawiki/2021/0/06/T--Linkoping_Sweden--EngCycleEng.png)

Figure 1.  An illustration of the engineering cycle.

## *Synechocystis sp. PCC 6803* Growth

At the beginning of our project, we decided to use the halophilic and photosynthetic cyanobacteria Synechocystis sp. PCC 6803.
Our reasoning was that it is a relatively well-established organism for synthetic biology. Synechocystis sp. PCC 6803 was also used by the SJTU BioX-Shanghai
2015 team for their project, which originally inspired us to choose desalination as the main goal of our project. During our growth curve experiments, we learned that Synechocystis sp. PCC 6803
was unable to grow for a long period of time in the non autoclaved sea- and brackish water. This led us to look at the samples under a microscope to try to figure out why our samples died.
To our surprise, we found other phototrophic organisms thriving in our samples (Figure 2). We concluded that some other phototrophs or combinations of phototrophs could be more suitable for desalination.
You can read more about the theorized phototrophs on our [results page.](https://2021.igem.org/Team%3ALinkoping_Sweden/Results)

![](https://static.igem.org/mediawiki/2021/d/d3/T--Linkoping_Sweden--Näyttökuva_2021-10-17_191247.jpg)

Figure 2. A.  Comparison of Synechocystis sp. PCC 6803 grown in BG-11 with an open atmosphere (left), BG-11 with a closed atmosphere (middle),
and non autoclaved brackish water from Gotland (right). **B.** Fluorescence microscope picture of the non autoclaved brackish water sample from Gotland.
Chlorophyll in the bacteria was excited with a wavelength of 480 nm. The circled organisms are identified as 1) Unknown, 2) Aphanizomenon.

From the same water samples, we were also able to spot some potential predators that were killing the Synechocystis cells. From literature,
we were able to possibly identify the predators as some kind of amoeba. We also found possible ways to genetically modify
Synechocystis to make it more resistant against the amoeba [1].
This would be an alternative way to improve our project and overcome the problem with cultivating bacteria in non autoclaved water.

We also had to adapt our cultivation setup for the Synechocystis sp. PCC 6803 during our experiments. Our original idea was to cultivate the bacteria as liquid cultures in Erlenmeyer
flasks sealed with aluminium foil. We soon discovered that our liquid cultures were not growing as dense as we hoped for. After some research and input from experts working with cyanobacteria,
such as Dr. Julie Zedler and Dr. Vamsi Moparthi, we decided to switch the aluminium foil to cotton plugs. This allows proper gas exchange into the flasks,
providing the bacteria with more CO2 which is needed for photosynthesis.

## Toxicity

When trying to amplify the halorhodopsin construct in Escherichia coli (strain DH5ɑ), we noticed that the colonies grew slower than expected and remained extremely small.
We made the same observations when halorhodopsin was transformed into Vibrio natriegens (strain Vmax), leading us to believe that halorhodopsin is toxic for both bacteria.
When analyzing a number of our halorhodopsin constructs through sequencing, the results showed that the sequence encoding halorhodopsin was not conserved in our plasmids.
The last part of the gene had been mutated in various positions in all products, causing a significantly shorter halorhodopsin to be expressed.
Based on these results, it is plausible that halorhodopsin is toxic to its host organism and that the sequence becomes mutated in order to preserve the survival of the host organism.
The possibly toxic effects of halorhodopsin is something that requires further research to be confirmed, but also something that should be taken into consideration in future research done using this ion pump.

## Constructs

Another difficulty came with ordering our constructs. The plan was to have the constructs we designed synthesized as a part of a plasmid but unfortunately,
the synthesis company failed to produce most of our constructs. This led us to order linear constructs with less complexity, meaning we removed the termination sequences,
and ligated these fragments into plasmids ourselves. As a result of cloning our constructs into plasmid ourselves, we needed to have a working system to select the colonies with the correct insert.
Our solution was to use pUC19, a plasmid commonly used for blue-white screening, but that we had not previously planned on using. The use of blue-white screening aided us in our laboratory work a lot. Furthermore, many failed ligation attempts led us to assume a new approach. In order to make our cloning more efficient, we decided to treat our plasmids with calf intestinal alkaline phosphatase (CIP).
CIP catalyzes the dephosphorylation of 5’ and 3’ ends of DNA phosphomonoesters [2]. The dephosphorylation prevents the self-ligation of the linearized plasmid.
This eliminates the possibility of getting colonies with a self-ligated plasmid with no intended insert.

---

#

[1] Simkovsky R, Daniels E, Tang K, Huynh S, Golden S, Brahamsha B. Impairment of O-antigen production confers resistance to grazing in a model amoeba-cyanobacterium predator-prey system. Proceedings of the National Academy of Sciences. 2012;109(41):16678-16683.

[2] New England BioLabs. Quick CIP [Internet]. New England BioLabs inc. [cited 17th October 2021]. Available from: <https://international.neb.com/products/m0525-quick-cip#Product%20Information>.

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Life depends on WATER but the reservoir depends on YOU.

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# Team:Linkoping Sweden/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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

# Team:Linkoping Sweden/Results

Team:Linkoping Sweden - 2021.igem.org

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* PROJECT
  + [Description](https://2021.igem.org/Team%3ALinkoping_Sweden/Description)
  + [Design](https://2021.igem.org/Team%3ALinkoping_Sweden/Design)
  + [Model](https://2021.igem.org/Team%3ALinkoping_Sweden/Model)
  + [Implementation](https://2021.igem.org/Team%3ALinkoping_Sweden/Implementation)
  + [Sustainabilty](https://2021.igem.org/Team%3ALinkoping_Sweden/Sustainable)
* LAB
  + [Results](https://2021.igem.org/Team%3ALinkoping_Sweden/Results)
  + [Improvement](https://2021.igem.org/Team%3ALinkoping_Sweden/Improvement)
  + [Contribution](https://2021.igem.org/Team%3ALinkoping_Sweden/Contribution)
  + [Engineering](https://2021.igem.org/Team%3ALinkoping_Sweden/Engineering)
  + [Notebook](https://2021.igem.org/Team%3ALinkoping_Sweden/Notebook)
  + [Protocols](https://2021.igem.org/Team%3ALinkoping_Sweden/Protocols)
  + [Safety](https://2021.igem.org/Team%3ALinkoping_Sweden/Safety)
* PARTS
  + [Parts Overview](https://2021.igem.org/Team%3ALinkoping_Sweden/Parts)
  + [Part Collection](https://2021.igem.org/Team%3ALinkoping_Sweden/Part_Collection)
  + [Basic Parts](https://2021.igem.org/Team%3ALinkoping_Sweden/Partsbasic)
  + [Composite Parts](https://2021.igem.org/Team%3ALinkoping_Sweden/Partscomposite)
  + [Vectors and Primers](https://2021.igem.org/Team%3ALinkoping_Sweden/vectorsprimers)
* PEOPLE
  + [Team Members](https://2021.igem.org/Team%3ALinkoping_Sweden/Team)
  + [Attributions](https://2021.igem.org/Team%3ALinkoping_Sweden/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3ALinkoping_Sweden/Collaborations)
  + [Sponsors](https://2021.igem.org/Template%3ALinkoping_Sweden/Spons)
  + [Team Album](https://2021.igem.org/Team%3ALinkoping_Sweden/Album)
* HUMAN PRACTICES
  + [Integrated Human Practices](https://2021.igem.org/Team%3ALinkoping_Sweden/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3ALinkoping_Sweden/Communication)
  + [Memory](https://2021.igem.org/Template%3ALinkoping_Sweden/Memory)
  + [Education](https://2021.igem.org/Team%3ALinkoping_Sweden/Education)
  + [Phototroph Handbook](https://2021.igem.org/Team%3ALinkoping_Sweden/Handbook)
* AWARDS
  + [Medal Criteria](https://2021.igem.org/Team%3ALinkoping_Sweden/Medal%20Criteria)

[Cultivation](#tab1)
![](https://static.igem.org/mediawiki/2021/6/64/T--Linkoping_Sweden--arrowDownButton.png)

[Growth](#subtab5)

[Predators](#subtab6)

[Symbiosis](#subtab7)

[Growth Model](#subtab8)

[Halorhodopsin](#tab5)

[References](#tab6)

## Results

## Cultivation of *Synechocystis sp. PCC 6803*

Successful and efficient growth of *Synechocystis sp. PCC 6803* is vital to enable testing of the parts involved in the CyaSalt desalination system and ultimately, to enable implementation of the desalination system in a seawater environment. Therefore, our project contained excessive wet-lab and dry-lab studies of the cultivation of *Synechocystis sp. PCC 6803* under different conditions.

Growth Conditions

As an effect of the slow growth rate of *Synechocystis sp. PCC 6803* (between 4-8 hours doubling time), optimizing its growth conditions was a crucial part of our project [1-3]. Therefore, analysis of the growth conditions for *Synechocystis sp. PCC 6803* constituted a large part of our wet-lab work. The growth media used for cultivation was BG-11. The composition of BG-11 can be found [here.](https://static.igem.org/mediawiki/2021/7/7b/T--Linkoping_Sweden--BG11Protocol.pdf) *Synechocystis sp. PCC 6803* is photosynthetic and hence it requires light to grow. Accordingly, the light source was one of the external factors evaluated. Supported by cultivation protocols obtained from two separate research groups working with *Synechocystis sp. PCC 6803*, the lights eventually used were of 340 Lumens and approximately 5500 Lux. Cultures were grown at a distance of 10 cm from the light source. Additional details about lamps used can be found [here.](https://static.igem.org/mediawiki/2021/e/e4/T--Linkoping_Sweden--CultivationLamp.pdf) A further aspect that was studied was the access to CO2 atmosphere provided to the culture flasks. After expert consultation, we realized that an open atmosphere would most likely increase the growth rate due to it providing the cultures with additional CO2. To obtain this, the culture flasks were sealed with cotton plugs which resulted in a substantial increase in culture density, as observed in Figure 1. Figure 1. gives a visual comparison of the growth in culture flasks sealed with cotton plugs and aluminium foil. The final growth setup used is illustrated in Figure 2.

![](https://static.igem.org/mediawiki/2021/b/bb/T--Linkoping_Sweden--CultureFlaskResults.jpeg)

![](https://static.igem.org/mediawiki/2021/6/63/T--Linkoping_Sweden--Incubator2.png)

Figure 2.  Illustration of the cultivation set-up used for the growth of *Synechocystis sp. PCC 6803*. The incubator used for plates and flasks is visualized. The Erlenmeyer flasks used are sealed with cotton plugs in order to enable open atmosphere access. The lamps were controlled by timers set to be turned on during 12 hours of the day and turned off during the remaining 12. The light intensities that were measured at different positions in the incubator are written in Lux.

Succeeding the growth optimization in BG-11, the growth of *Synechocystis sp. PCC 6803* was studied under various other conditions. Since our desalination system ultimately relies on the phototrophs growing in seawater, we collected several water samples to test the growth. Water samples with a salt percentage of approximately 1% were retrieved from the Baltic Sea. Furthermore, samples were collected from the North Sea with a salinity of approximately 3%. The graph in Figure 3. illustrates the results obtained from growing *Synechocystis sp. PCC 6803* in these samples, both autoclaved and non-autoclaved, in addition to in BG11.

![](https://static.igem.org/mediawiki/2021/4/4d/T--Linkoping_Sweden--graphResults.jpeg)

**Figure 3.** Growth curve for *Synechocystis sp. PCC 6803*. The X-axis shows the time in days from seeding and the y-axis shows the measured optical density at 544 nm. The media which *Synechocystis sp. PCC 6803* used is displayed in the legend to the right in the figure. Seawater samples with ~3% salinity, retrieved from the North Sea in Gothenburg, Sweden are abbreviated GBG and brackish seawater samples retrieved from the Baltic Sea on Gotland, Sweden have a salinity of ~1%. The smaller graph included in the figure shows the optical density in the BG-11, GBG not autoclaved and Gotland not autoclaved cultures during the first 6 days. This was done to highlight that *Synechocystis sp. PCC 6803* started dying in the not autoclaved water during that time.

The graph displays substantial growth in the BG-11 media. The growth in the autoclaved seawater also exhibits a rather steady growth curve, although at a considerably slower rate than in BG-11. The cultures grown in autoclaved seawater had a much lower optical density of *Synechocystis sp. PCC 6803* compared to the ones grown in BG-11. The cells also exhibited a yellow color as observed in Figure 1., most likely due to deficiency of a nutrient. The results that correlate best with real-world implementation are from the non-autoclaved seawater cultures. In these samples, the graph shows a slight growth initially. However, after only 4 days, the *Synechocystis* cultures in non-autoclaved seawater began to die. This was further visually observed by the green color of the cultures fading.
 Moreover, samples from the dying cultures were looked at under an epifluorescence microscope. Here, observations further suggested that the *Synechocystis sp. PCC 6803* were dying. Figures 4A and 4B show images of healthy *Synechocystis sp. PCC 6803* cultures taken with an epifluorescence microscope whereas Figure 4C. shows a dying culture. Figure 4C. shows that no fluorescence was obtained from the dying *Synechocystis sp. PCC 6803* cultures.

![](https://static.igem.org/mediawiki/2021/e/ee/T--Linkoping_Sweden--MicImages4.png)

**Figure 4.** Images showing **A)** A microscopic image (40x) of a healthy *Synechocystis sp. PCC 6803* culture excited at 535 nm. **B)** A spectral microscopic image (40x) of a healthy *Synechocystis sp. PCC 6803* culture excited at 560 nm. **C)** A spectral microscopic image (40x) of a dead *Synechocystis sp. PCC 6803* culture excited at 560 nm.

Predators Native to Seawater

Additional microscopic observations of the non-autoclaved seawater cultures presented a possible reason for *Synechocystis sp. PCC 6803* dying. Organisms, shown in Figure 5., possibly preying on *Synechocystis sp. PCC 6803* were observed and possibly identified. These observations lead us to speculate that predators living in the seawater, possibly amoebae, have caused the death of *Synechocystis sp. PCC 6803*. Previous studies have suggested that a potential solution to amoebas preying on cyanobacteria is to impair the O-antigen production in *Synechocystis sp. PCC 6803*. This would result in them not being recognized by the amoebae. However, a potential risk to this solution is that it motivates evolution in the amoeba that enables them to identify the modified cyanobacteria [4].

![](https://static.igem.org/mediawiki/2021/d/d1/T--Linkoping_Sweden--PredatorsResults.png)

**Figure 5.** Bright-field images (40x) of the potential eukaryotic predators found in non-autoclaved seawater samples retrieved from Gotland (salinity ~1%) with *Synechocystis sp. PCC 6803*. The organisms in the image did not exhibit any fluorescence.

Symbiotic Phototrophic Organisms Native to Seawater

Another suggestion is to use another phototrophic organism that is not targeted by the predator. Evidence encouraging this possibility is that various phototrophic organisms were still thriving in symbiosis in the non-autoclaved seawater after *Synechocystis sp. PCC 6803* had died (appearing after 25 days). These phototrophic organisms were noticed through microscopic studies and are shown in Figure 6.

![](https://static.igem.org/mediawiki/2021/9/92/T--Linkoping_Sweden--MicImages6.png)

**Figure 6.** Native phototrophs that grew in non-autoclaved seawater from Gotland (**A-D**) and Gothenburg (**E, F**). The phototrophs were identified as (1) Unknown, (2) Aphanizomenon, (3) Nitzschia, (4) Unknown, (5) Unknown, (6) Unknown, (7) Unknown, (8) Unknown, (9-10) Nostoc sp or Nodularia, (11) Unknown, (12) Unknown, (13) Gloeocapsa, (14) Woronichinia sp. All the phototrophs were identified by comparing morphology [5]. The images were generated with an epifluorescence microscope (Leica Spectraview). Excitation wavelength for the corresponding picture (**A**) 480 nm, (**B**) 405 nm, (**C-D**) 436 nm, (**E**) 480 nm, (**F**) 350 nm.

Some of the observed organisms were identified by comparing morphology. The identified species could be Aphanizomenon, Nitzschia, Nostoc sp. or Nodularia, Gloeocapsa and Woronichinia sp. [5]. Since these phototrophic organisms exhibit superior growth in seawater compared to *Synechocystis sp. PCC 6803*, they could be better suited to implement the CyaSalt desalination system in. Especially Aphanizomenon and Nodularia could work for desalination of Baltic seawater, as they normally bloom in large quantities in the Baltic Sea [6]. For future work, one option is to modify multiple of the observed phototrophic organisms to obtain a symbiotic desalinating system. Another option is to continue with one of the organisms and attempt to implement our desalination system in it. Conclusively, the best approach needs to be determined by further studies.

Growth Prediction by the Model

A model was created to enable growth predictions of phototrophic organisms. Experimental data obtained from *Synechocystis sp. PCC 6803* cultivation under various conditions was applied to the model and predicted growth curves were generated. The model is adaptable so that the growth of any phototrophic organism, wild-type or modified, can be predicted. Read more about modelling results and how it has affected our project [here.](https://2021.igem.org/Team%3ALinkoping_Sweden/Model)

## Expression of Halorhodopsin

The part BBa\_K3892001, coding for the ion pump Halorhodopsin, were ordered from Integrated DNA Technologies (IDT) cloned into the plasmid pUCIDT-Amp. However, they experienced problems with producing it and were ultimately only able to produce a low amount of product. After receiving this product, it was transformed into *E. coli* (strain DH5 alpha) as well as *V. natriegens* (strain Vmax). However, the resulting colonies were small and they also grew slower than anticipated. When sending a number of plasmid preps for sequencing, the results showed that the sequence encoding Halorhodopsin was not conserved in our plasmids. The last part of the gene had been mutated at various positions in all products, causing a significantly shorter Halorhodopsin to be expressed. Based on these results, it is plausible that Halorhodopsin is toxic to its host organism and that the sequence mutates in order to preserve the survival of the host organism. The possibly toxic effect of Halorhodopsin is something that requires further research to be confirmed, but also something that should be taken into consideration in future research done with this ion pump.

---

#

[1] Cordara A, Re A, Pagliano C, Van Alphen P, Pirone R, Saracco G, Branco Dos Santos F, Hellingwerf K, Vasile N. Analysis of the light intensity dependence of the growth of *Synechocystis* and of the light distribution in a photobioreactor energized by 635 nm light. PeerJ. 2018 Jul 27;6:e5256. doi: 10.7717/peerj.5256. PMID: 30065870; PMCID: PMC6065478.

[2] van Alphen P, Abedini Najafabadi H, Branco Dos Santos F, Hellingwerf KJ. Increasing the Photoautotrophic Growth Rate of *Synechocystis sp. PCC 6803* by Identifying the Limitations of Its Cultivation. Biotechnol J. 2018 Aug;13(8):e1700764. doi: 10.1002/biot.201700764. Epub 2018 May 2. PMID: 29577667.

[3] Zavřel T, Sinetova MA, Búzová D, Literáková P, Červený J. Characterization of a model cyanobacterium *Synechocystis sp: PCC 6803* autotrophic growth in a flat-panel photobioreactor. Eng Life Sci. 2015;15(1):122–32. doi: 10.1002/elsc.201300165.

[4] Simkovsky R, Daniels EF, Tang K, Huynh SC, Golden SS, Brahamsha B. Impairment of O-antigen production confers resistance to grazing in a model amoeba-cyanobacterium predator-prey system. Proc Natl Acad Sci U S A. 2012;109(41):16678–83. doi: 10.1073/pnas.1214904109.

[5] Wiśniewska K, Lewandowska AU, Śliwińska-Wilczewska S. The importance of cyanobacteria and microalgae present in aerosols to human health and the environment - Review study. Environ Int. 2019 Oct;131:104964. doi: 10.1016/j.envint.2019.104964. Epub 2019 Jul 24. PMID: 31351382.

[6] Walve J, Larsson U. Blooms of Baltic Sea Aphanizomenon sp. (Cyanobacteria) collapse after internal phosphorus depletion. Aquat Microb Ecol. 2007;49(1):57–69.

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