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	Be Basic	Synenergene

LactoAid – A smart bandage for burn wounds

Finnish by courtesy of the university of Helsinki

French by courtesy of the university of Paris Saclay

Norwegian by courtesy of the university of Oslo

Polish by courtesy of the university of Warsaw

Portuguese by courtesy of the university of São Paulo

Infections caused by Staphylococcus aureus and Pseudomonas aeruginosa often pose problems for burn wound treatments. We developed a new kind of bandage that prevents these infections and reduces the use of antibiotics, thereby lowering the risk of developing antibiotic resistance. The bandage consists of a hydrogel that contains genetically engineered Lactococcus lactis with nutrients. The engineered strain of L. lactis detects the quorum sensing molecules of the two pathogens in the wound and subsequently produces the antimicrobial nisin as well as some other Infection-Preventing-Molecules (IPMs). These IPMs are the anti-biofilm protein Dispersin B and the quorum quenching protein AHLase. The gel is placed between two layers, a top layer to allow diffusion of gases and a bottom layer to contain the bacteria within the bandage. Hydrating the gel by breaking adjacent water pockets initiates the growth of the bacteria, thereby activating the bandage.

LactoAid-给灼伤一个聪明的敷料

金黄色葡萄球菌和绿脓杆菌经常导致灼伤伤口感染,影响治疗。本研究研制了一种敷料,既可以预防此类感染,又能减少抗生素的使用,因此降低抗生素抗药性的发生。此敷料带有一种水凝胶,里面含有转基因乳酸乳球菌和它所需要的营养成分。转基因的乳酸乳球菌能够探测到灼伤伤口里两种病原体的群体感应分子,并发出乳酸链球菌肽以及另外两种抗感染分子 (Infection Preventing Molecules, IPMs)。这两种抗感染分子分别是分散蛋白(Dispersin B)和群体感应淬灭酶酰基高丝氨酸内酯酶。水凝胶夹在两层中间,上层使敷料透气,下层控制敷料里的细菌。通过把邻近的水包穿破,使水凝胶产生水合反应,因而使细菌开始生长,这样就可以激活此敷料了。

See us on the Dutch national news!

LactoAid - Een slim verband voor brandwonden

Infecties door Staphylococcus aureus en Pseudomonas aeruginosa zorgen vaak voor complicaties bij de behandeling van brandwonden. We hebben een nieuw soort verband ontwikkeld dat deze infecties tegengaat en het gebruik van antibiotica terugdringt waardoor het risico op de ontwikkeling van antibiotica resistentie verminderd wordt. Het verband bestaat uit een hydrogel die genetisch gemodificeerde Lactococcus lactis bevat met nutriënten. Deze gemodificeerde stam van L. lactis detecteert 'quorum sensing' moleculen van de twee pathogenen in de wond en produceert vervolgens zowel het antimicrobiële nisine als enkele andere Infection-Preventing-Molecules (IPMs). Deze IPMs zijn het anti-biofilm eiwit Dispersin B en het quorum quenching eiwit AHLase. The gel zit tussen twee lagen, een bovenlaag die de diffusie van gassen accomodeert en een onderlaag om de bacteriën in het verband vast te houden. De groei van bacteriën begint als de gel bevochtigd wordt door naastliggende waterzakjes te breken, zodat het verband geactiveerd wordt.

LactoAid/LaktoApu Älyside palovammoille

Staphylococcus aureus ja Pseudomonas aeruginosa bakteerien infektiot aiheuttavat usein ongelmia palovammojen hoidossa. Me kehitimme uudenlaisen siteen, joka ehkäisee näitä infektioita ja vähentää antibioottien käyttöä, ja siten vähentää antibioottiresistenssin kehittymisen riskiä. Siteessä on ravinteita sisältävää hydrogeeliä, jossa on geneettisesti paranneltua Lactococcus lactis bakteeria. Tämä paranneltu kanta tunnistaa näiden kahden patogeenin erittämiä viestimolekyylejä haavassa, minkä seurauksena se tuottaa antimikrobista nisiiniä sekä infektiota estäviä molekyylejä, biofilminestäjäproteiini Dispersiini B:tä sekä patogeenien viestintää tukahduttavaa AHLaasia. Geeli sijaitsee kahden kerroksen välissä; päällimmäinen kerros hengittää ja alempi kerros estää bakteerien pääsyn geeliltä iholle. Kun siteessä olevat vesitaskut puhkaisee, geeli kostuu ja käynnistää bakteerien kasvun, jolloin älyside aktivoituu.

LactoAid - Un pansement intelligent pour des brûlures.

Les infections causées par Staphylococcus aureus et Pseudomonas aeruginosa posent souvent problème dans le traitement des brûlures. Nous avons développé un nouveau type de bandage qui prévient ces infections et permet de réduire l’utilisation d’antibiotiques et donc le développement de résistance à ces antibiotiques. Le pansement consiste en un hydrogel qui contient des Lactococcus lactis génétiquement modifiées ainsi qu’une source de nutriments. L.lactis a été génétiquement modifiée pour détecter les molécules de quorum sensing des deux pathogènes dans la plaie et ensuite sécréter la nisin, un antimicrobien et deux autres molécules préventives d'infection, la Disperine B -une protéine anti-biofilm- et l'AHLase impliquée dans le quorum quenching (dégradation du biofilm).Le gel est placé entre deux couches, une couche supérieure pour permettre la diffusion des gaz et une couche inférieure pour contenir les bactéries à l'intérieur du bandage. L’hydratation du gel - en cassant les poches d'eau adjacentes - initie la croissance des bactéries, ce qui active le bandage.

LactoAid - Un pansement intelligent pour des brûlures.

स्टेफाईलोकॉकस औरीयस और सूडोमोनस एरोगिनोसा की वजह से संक्रमण अक्सर जला घाव उपचार के लिए समस्या पैदा कऱता है। हमने एक नए प्रकार की पट्टी का विकास किया है, जो संक्रमणों से बचाता है और एंटीबायोटिक दवाओं के उपयोग को कम कर देता है और इस तरह एंटीबायोटिक प्रतिरोध विकसित होने का खतरा कम कर देता है | पट्टी हाइड्रोजेल की होती है जिसमे पोषक तत्वों के साथ आनुवंशिक रूप से विकसित लक्टोकॉकस लैक्टिस होता है। जिसमें एक एल लैक्टिस में उभरा तनाव घाव में दो रोगजनकों का कोरम संवेदन अणुओं का पता लगाता है और बाद में रोगाणुरोधी नाइसिन साथ ही कुछ अन्य संक्रमण के रोकथाम अणुओं (आईपीएमएस) को पैदा करता है । ये आईपीएमएस विरोधी जैव झिल्ली प्रोटीन डिस्पेरसिन बी कोरम शमन प्रोटीन एएचएलऐस हैं , जेल दो परतों के बीच रखा गया है, शीर्ष परत गैसों के प्रसार और नीचे की परत पट्टी के भीतर जीवाणु को रोकने के लिए रखा गया है ।, आसन्न पानी जेब टूटने से जेल पोषित होता है जिससे जीवाणु की वृद्धि होती है और पट्टी सक्रिय होती है

LactoAid – En lur bandage for brannsår

Innfeksjoner forårsaket av Staphylococcus aureus og Pseudomonas aeruginosa medfører ofte problemer ved behandling av brannskader. Vi har utviklet en ny type bandasje som hindrer disse innfeksjonene og reduserer bruken av antibiotika og dermed også risikoen for å utvikle antibiotikaresistens. Bandasjen er en hydrogel med genetisk modifiserte Lactococcus lactis og næringsstoffer. L. Lactis responderer på quorom sensing molekyler fra de to patogenene med å produsere antimikrobielt nisin sammen med andre infeksjonshemmende molekyler(IPM). Blant disse er anti-biofilm proteinet Dispersin B og quorum quenching proteinet AHLase. Gelen er plassert mellom to lag, et lag på toppen som tillater diffusjon av gasser og et lag på bånn som inneholder bandasjens bakterier. Ved å knuse små vannlommer fuktes gelen og dette initierer bakterieveksten, således aktiveres bandasjen.

LactoAid – sprytny opatrunek na oparzenia

Infekcje spowodowane przez Staphylococcus aureus i Pseudomonas aeruginosa często stanowią problem przy leczeniu ran pooparzeniowych. Zaprojektowaliśmy nowy rodzaj opatrunku, który chroni przed takimi infekcjami i ogranicza stosowanie antybiotyków, co obniża ryzyko rozwoju odporności na antybiotyki u bakterii. Na opatrunek składa się hydrożel zawierający genetycznie zmodyfikowany szczep Lactococcus lactis wraz z pożywką. Zmodyfikowany szczep L. lactis rozpoznaje cząsteczki wytwarzane przez bakterie patogenne podczas wyczuwania liczebności (quorum sensing) i następnie wytwarza antybakteryjną nizynę wraz z innymi cząsteczkami chroniącymi przed infekcją (Infection- Preventing Molecules, IPMs). Do tych innych cząsteczek należą białko anty-biofilmowe Dispersin B i białko zmniejszające liczebność – AHLiaza. Żel znajduje się pomiędzy dwoma warstwami: górna warstwa umożliwia dyfuzję gazów, a dolna zawiera bakterie umieszczone wewnątrz opatrunku. Uwodnienie żelu poprzez łamanie sąsiednich kieszonek wodnych inicjuje wzrost bakterii, w ten sposób aktywując działanie opatrunku.

LactoAid – Um curativo inteligente para queimaduras

Infecções causadas por Staphylococcus aureus e Pseudomonas aerugionosa são problemas recorrentes no tratamento de queimaduras. Nós desenvolvemos um novo tipo de curativo que previne essas infecções e reduz o uso de antibióticos, diminuindo o risco de desenvolvimento de resistência a estes medicamentos. O curativo consiste em um hidrogel que contém Lactococcus lactis geneticamente modificadas com nutrientes. A cepa modificada de L. Lactis detecta as moléculas quorum sensing dos dois patógenos na ferida e subsequentemente produz o antimicrobial nisina, como também outras moléculas preventivas de infecção (IPMs). Estas IPMs são as proteínas anti-biofilme Dispersina B e a proteína de quorum quenching AHLase. O gel é posicionado entre duas camadas, uma camada superior para permitir a difusão de gases e uma camada inferior para conter as bactérias no curativo. A hidratação do gel através da quebra de bolsos de água adjacentes inicia o crescimento da bactéria, ativando assim o curativo.

Figure 1: The lactoAid is a bandage containing Lactococcus lactis

The iGEM Groningen team 2014 is creating a bandage containing Lactococcus lactis that produces nisin to destroy gram-positive bacteria. The bandage consists of a top layer that permits air to flow through and keeps the bandage clean, a middle layer of hydrogel that holds the L. lactis, and a bottom layer of higher density hydrogel that allows proteins to pass through but contains the L. lactis to the middle layer. Additionally we wll introduce the excretion of AHL lactonase and Dispersin B, compounds that disrupt quorum sensing and biofilm formation respectively of Staphylococcus aureus and Pseudomonas aeruginosa. To take it even further, the bandage will detect quorum sensing molecules produced by S. aureus and P. aeruginosa. Thus the bandage will excrete nisin and Dispersin B when S. aureus is present and AHL lactonase when P. aeruginosa is present.

Infections in burn wounds are currently only treatable with antibiotics. An increase in antibiotic resistance makes it harder every day to fight these bacteria. In our system L. lactis produces a so-called lantibiotic, nisin. Nisin is effective against a group of gram-positive bacteria such as Staphylococcus aureus and resistance against nisin is hardly ever found and if found, the resistance does not last.

Beside nisin the L. lactis will be able to produce the infection preventing molecules (IPMs) AiiA and DspB. AiiA will disrupt the communication mechanism of the harmful bacteria, this way the bacteria will not cause any trouble because it 'thinks' it is alone. DspB is a molecule that prevents the harmful bacteria to form a layer (biofilm) on the wound. Additionally we want to try to make the bandage 'active' (producing nisin, DspB and AiiA) only when harmful bacteria are present in the wound. The bandage targets Staphyolococcus aureus and Pseudomonas aeruginosa specifically, two bactefria that are a problem in burn wound centres.

Figure 2: Quorum sensing molecules pass from the wound to the lactococcus lactis, in turn IPMs pass from the bandage into the wound.

The design of the bandage is important as well. L. lactis should not be able to get out of the bandage, but the IPMs should be able to reach the wound (See figure 2). Besides containing L. lactis the bandage should allow sufficient oxygen to reach the wound.

Finally, the whole package needs to be able to be stored for quite a while and still work. Therefore L. lactis will be stored as a powder and can be activated with water when the bandage is needed.

We are also investigating the possibilities of having L. lactis produce growth factors to aid in wound healing and to link the detection to the production of a chromoprotein to show when the bandage detects harmful bacteria.

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Because it is important for GMOs to be contained to where it is used we had to start by thinking how we could keep our bacteria contained inside the bandage, while still making sure it can perform its functions. We had many discussions about all the parts to make sure they meet our needs

Since we were already sure we were working with Lactococcus lactis our needs were clear from the start.

Very soon we thought of a gel like material to put our bacteria in with a top and bottom membrane to keep everything inside. The top membrane needed to allow for the diffusion of gases to maintain oxygen levels in the wound, while the bottom needed to allow all the peptides and proteins to diffuse outside to kill or hamper our target bacteria while immobilising our bacterium.

Design requirements:

Materials:

The materials should not be toxic to humans or bacteria.

The materials should be permeable to gasses

It should be durable and preferably inexpensive

Since it is a bandage it should not break too easily and it should be flexible.

Appearance

We prefer the bandage to be transparent so that you can look through it and judge whether it should be changed or it can stay on for another day.

Safety

since we use the bandage on a wound we really had to make sure our bacteria stays inside the gel. If the bacteria does not stay in the gel this can make another infection happen.

We will give the bottom layer a pore size where Lactococcus lactis stays finside while the other peptides and proteins diffuse inside and outside the gel.

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Synenergene project
Synenergene asked us to make an implementation scenario for our project. Below you can read the desired future 'history' of the LactoAid.

Application scenario
We are now in the year 2028, the world population has increased to over 8 billion people, putting an ever increasing pressure on healthcare to maintain a good health for all. After facing major problems regarding antibiotic resistance in the early tens, a different kind of product was developed and marketed that could regulate the use of antibiotics and prevented the development of any resistant strains. Due to a well-defined implementation strategy of this technology in society, and by creating specific safeguards in the product, the rise of resistant strains has been brought to a halt. This story outlines the history of LactoAid, explaining how this product came to be and why it is such a success in modern healthcare.

It all started with the development of the concept by a group of students: to have a bandage that only produces antimicrobial compounds when needed, contrary to applying loads of antimicrobial compounds in advance (thus increasing the risk of developing resistant strains). A bandage that gradually releases antibiotics was one of the first ideas. Although this might prolong the use of a single bandage, thereby reducing the risk of infection, it still applied antibiotics in a preventive way. Also, electronic systems could be a carrier for this concept, but these were quickly discarded due to their limited applicability on the relatively small burn wounds, and given the high manufacturing costs involved. After this, it was argued that a perfect host to hold such a system would be a single-celled organism; a bacterium. Bacteria are small, easy to produce, and can be engineered to fulfil specific functions. However, the field of synthetic biology was just starting to emerge. Not much was known about the implications of messing around with the DNA of living organisms, resulting in societal hesitance to accept this new technology. To reduce the scope of the project, it was decided to take burn wounds as a case-study for such a concept. At a later stage in the product development, the bandage was also developed to fit other types of wounds.

The initial arguments against the implementation of GMO bacteria in a healthcare environment were mainly related to ethical and societal issues. Questions like; do the bacteria go into the wound? What is the risk of the bacteria getting out of the bandage into the environment? Is there a safeguard in case the bacteria leak out of the bandage? What are the chances of the bacteria mutating into a harmful pathogen? And many more. After these issues were addressed in the design of the bandage, as well as in the genetic design of the organism itself, the further development of the product was initiated. First of all, investors and strategic partners needed to be found to allow the full-scale deployment of this bandage. These were particularly important with regard to the scale of this project and to acquire funding (see timeline). These partners and investors were found by contacting research institutes and global health organizations (WHO, Red Cross).

The full programme – from the concept generation to the product launch – was organized as a joint venture. Many parties from different industries were involved, and these were led by the group of students that started the project. It must be noted the large pharmaceutical companies (Bayer, GlaxoSmithKline, Astrazeneca) were hesitant in the initial stage of product development, but contributed at a later stage when the demand for novel antibiotic moderators became crystal clear. A list of all parties involved in the final stage of the product development:

Healthcare related organizations

Hospitals
Burn wound centers
WHO
International Red Cross and Red Half Moon movement
Health insurance companies
Big pharma (at a later stage)

Consumer organizations

First aid kits including infection detection systems
The bandage to be sold in pharmacies

The final product development, as described in the timeline, started in 2015 by generating different designs and testing their techno-economic feasibility. By 2018 the primary research and development was finalized, and the functionality of the best 5 designs could be tested in the lab (e.g. animal experiments) in the preclinical stage. The clinical trials were held between 2020-2026, after which all documentation was submitted to the EMA and FDA for approval regarding safety, risk and quality of the bandage. In 2027, both the FDA and EMA granted their approval for the LactoAid.

It was a difficult road to market, especially regarding regulatory and safety issues, but finally the worldwide demand of this type of bandage was sufficient to drive the final development and make it through trials. Nowadays, the bandage is used in hospitals all over the world to treat open wounds. A consortium of companies and research institutes has been tasked to oversee the production and regulate the use of this bandage. This consortium is led by the WHO. There is no problem anymore with multi-resistant strains, and wounds can easily be kept clean throughout a patient’s stay. There is even talk of a LactoAid 2.0 (now in trials) that is genetically tweaked to produce growth factors. These are substances that stimulate cell growth. The result will be a super bandage that has the ability to heal certain wounds even faster.

The application of LactoAid is best explained through an exemplary patient that enters a regular hospital:

A patient enters the hospital, is heavily burned all over his/her body with 2nd and 3rd degree burn wounds.
The wounds are cleaned and then left to rest for three days to assess the severity of the wounds.
During these three days, the wound is protected by LactoAid.
After three days the wounds are found to be severe, and a skin transplant is needed.
The wound is rinsed again, and the skin transplant takes place. This needs to be done in multiple sessions due to the heavy impact the operation has on the body. This leads to a lot of time in which the wounds need to be kept clean. Therefore, LactoAid is again applied to the wound in the time between the operations.
As a result; A) patients suffer less because there is no free air flow over the wounds, B) the wound is protected by the bandage from environmental pathogens, C) pathogens already in the wound are killed by the bandage, reducing the number of times the bandage needs to be replaced, D) wounds are healed quicker, cutting costs.
After every use (3-4 days), the bandage is deactivated and all modified bacteria are killed. Two systems are used to ensure no modified bacteria survive: A) after 3-4 days all nutrients are gone, B) a kill switch is activated by releasing an inducing compound within the bandage.

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Towards an application scenario

Interview burn centre at the Martini-hospital in Groningen, the Netherlands

Nearly halfway through our iGEM-adventure of creating an infection-detection-protection dressing for (burn) wounds, we got the opportunity to talk with some experts in the field. Of course, creating a ‘genetically engineered machine’ is what we do, but we want to explore the application further. Having a talk with people who could eventually use our concept in real-life was therefore essential; if there is no need for this type of dressing, then there is no need to develop it! Marianne Nieuwenhuis is the head of clinical research for the three cooperating burn centres in the Netherlands and is based at the burn centre of the Martini hospital in Groningen. Jakob Hiddingh is also affiliated to the burn centre and is working on clinical research trials. Whenever a researcher is not around, Jakob takes over the responsibilities, making him an expert on the practical side of clinical research trials.

Can you explain something about the burn centres? How do they work? What kind of research should we imagine when you talk about clinical research trials?

‘In the Netherlands there are about 600-800 recordings (annually) of patients in the three burn -centres; Beverwijk, Rotterdam and Groningen. Rotterdam is the largest of these three. The time patients stay at the centre varies from one day to several months. We do research in everything ranging from psychological research to physical issues like itching. On average, patients remain at the centre for 14 days, but as said, this varies a lot depending on the patient and severity of the injury.’

As you may know, we are developing a dressing that is aimed at protecting burn wounds, specifically to detect an infection of S. aureus or P. aeruginosa. Do many problems with infections occur when treating burn wound patients? How do you handle these?

‘There is a clear distinction between an infection and a wound that has some pathogen (harmful bacteria) in it; a colonization. Colonization is when the pathogen is there, but you do not get ill from the presence of these bacteria. When you talk about an infection, many of the classical features occur and these are very harmful for the healing process. In this centre, we typically take a sample of all the wounds once a week to see if there are any harmful bacteria on them. We also take a sample of the throat and nose. ‘

Groningen, and the Netherlands in general, does not have many problematic infections. How is this problem of infection prevention taken care of in the rest of the world?

‘It’s hard to compare numbers because every centre and hospital has its own way of detecting infections. There is a large range of pathogens, but especially S. aureus and P. aeruginosa are the pathogens that cause most problems.’

If we understood well, Flammacerium (a cream-like substance) is used mostly to keep the wound clean and improve wound healing. How does this treatment work and is it used in other parts of the world?

‘Flammacerium consists of two components, the silver sulphadiazine (also known as Flammazine), and cerium nitrate. The silver sulphadiazine keeps the wound clean, but a serious downside is that it induces hypergranulation after a while (the overproduction of tissue). People have been looking for an alternative for quite some time, but so far nothing realy superior is on the market yet. We believe that the cerium nitrate does have a positive impact on the treatment of burn wounds, therefore we keep using Flammacerium. In other parts of the world this treatment method is used to a lesser extent.’

For our dressing, we would like to think a step further than only developing it and then putting the prototype in the fridge. What are the main hurdles to be taken when developing this dressing for burn patients?

‘Keep in mind that for every minor step you want to do when dealing with a clinical research trial, you should write a protocol and all actions should be cleared by the medical-ethical commission. In the Netherlands, this is the case for every WMO-research (research involving humans). For the testing of a new product, such as your dressing, you would need to fill out many forms involving a complete description, how it works, what could go wrong, what is the goal and added value etc. etc. .’

As we are speaking about regulation issues regarding new products, what do you think of our idea once it has gone through all the clinical trials and been proven to work, say in 10 years? Do you see the relevance of having such a dressing? Do you foresee any ethical issues that might arise in the practical application of GMO’s in healthcare?

‘The funny thing is, I never heard of synthetic biology before you came along. I did not realize this project would involve any moral dilemmas that needed to be so seriously addressed. I just thought: that’s great, they are “playing around” to make bacteria do a specific task! As long as the added value is clear, in other words, if it helps the patients to get better, there will be not so much resistance against it. It should be very well regulated though, but this is already under debate. I understand that genetically modified organisms are a bit difficult to use and there is some resistance, but to me this project seems very worthwhile.’

Do you have any advice for us that could help us in the development of our dressing?

‘We are enthusiastic about your idea and think that this is an innovative approach to treating wounds. The idea I like so much is that it goes beyond normal theoretical research, but you are actually making it. It is a quite appealing concept. There is also possibly less resistance as you are trying to make a better treatment for patients with burn wounds. You should have a talk with our medical microbiologist and medical director!’

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TEDxTwenteU – Ideas worth spreading. In the spirit of TED.com, a TEDx event will be organized at the 10th of October at the University of Twente. At a TEDx event, speakers will talk about their vision, ideas and inspirations in order to inspire the attendees and viewers. We will also be speaking at TEDx, and we hope to inspire many minds to think outside the box when tackling the problem of infections.

Get ready to be inspired at http://www.tedxtwenteu.nl/ ...!

Falling walls is a yearly science event in Berlin where researchers can tell how their walls breaks the walls of humanity’s big problems. The day before the Falling walls conference, the falling walls lab is organized, where young researchers can present their work. Speakers get three minutes to tell the audience about their project, and convince them of its importance.
Andries went to the qualifying round in Groningen where he won us the first prize. After the iGEM finale in Boston, he will fly back to Amsterdam, take the train to Berlin, and tell a large international audience about our bandage on November 8./p>

Sorry, nothing here yet, but check out our discovery festival piece!

September the 26th the iGEM team 2014 will present at Discovery festival Amsterdam 2014.

Discovery festival is, as the name says, a festival celebrating science and music. It was started in Amsterdam in 2006 because the founders of Discovery festival observed a lack of events combining these two cornerstones of civilisation.

Dicoveryfestival was great!

Due to the 400th anniversary of the University of Groningen, ScienceLinX organized several expositions throughout Groningen, and one spectacular evening, with all kinds of artists and events. The evening, better known as the “Night of Arts and Sciences” took place at the 24th of May.
We were lucky enough to get a spot in the Der Aa-Kerk in the centre of the city during the “Night of Arts and Sciences”. Throughout the exposition, the project of the iGEM 2012 team of Groningen was shown to warm the public. At the night itself, we introduced synthetic biology, the iGEM competition, and our bandage to the public. People were able to win a small consumable prize if they answered a sufficient amount of question of our quiz correctly. All in all a successful night!

12-06-2014	The university newpaper places an article on this year's iGEM team on their website: iGEM team wil superverband maken
17-09-2014	Andries of our team won the national preliminaries of falling walls lab: RUG-student Andries de Vries wint Nederlandse voorronde Falling Walls
24-09-2014	Unifocus reports on the project through a short video clip: Unifocus website
24-09-2014	ANP (Algemeen Nederlands Persbureau or General Dutch Press) picked up our story. It is presented on sites like nu.nl: Studenten willen brandwonden genezen met yoghurtbacterieën Other sites like Hart van Nederland placed the report.
26-09-2014	The university paper reports again on our project: Deadline approaches for iGEM team.
26-09-2014	The Dutch biological society reports on all the dutch iGEM teams in their newsletter: iGEM: Teams full of confidence at work

Sorry, nothing here yet!

What is Synthetic biology?

Figure 1: A plasmid (A) with an antibiotics resistance gene (in green) can be cut by restriction enzymes (dark red) (B). A new piece of DNA (bright red) can be insterted into the gap (C). The insertion is 'glued' into place by ligases (D). We then have our product to insert into bacteria (E).

Synthetic biology is the science of constructing biological systems and devices through application of biological knowledge and synthetic chemistry. In the case of the iGEM competition, the basic idea is to make relatively simple genetic constructs and apply those constructs to a task through bacteria. We go through a simplified example here. The reality has a lot more steps but the broad strokes remain the same.

Bacteria have their own tools with which they can read genetic code, and translate that code into proteins; the molecules that perform nearly all functions within a living cell. We can use these tools when we insert custom-made DNA sequences into the bacteria in the form of plasmids. Plasmids are 'rings' of DNA, which bacteria can pick up and use. In nature, bacteria share plasmids to gain new functions.

Before we have our plasmid with the desired DNA sequence, we will have to design it. Usually, one starts with a 'backbone' plasmid, and the desired piece of DNA. Such plasmids are often isolated from bacteria found in nature and contain a DNA sequence with an ability that makes the bacteria able to survive conditions that other bacteria cannot. The most common are antibiotic resistance genes.

If we have a backbone, we can insert a sequence of DNA into the plasmid by cutting it with restriction enzymes, and pasting in the DNA sequence with DNA ligases. Restriction enzymes are molecules that can recognize very specific parts of DNA, called restriction sites, and cut them (see figure 1: A, B). DNA ligases are molecules that can 'glue' together two large molecules, such as two ends of DNA.

Restriction enzymes can be used to cut a desired piece of DNA from another bacteria, to transplant into our backbone. Then the backbone is cut, the desired seqeunce is inserted and our plasmid is ready to be inserted into a bacteria (see figure 1: C, D, E).

When we insert our plasmid, the bacteria will read the DNA, and produce the product. We can abuse the bacteria's own system to make bacteria produce molecules like DspB, a molecule which is already used in nature by bacteria that make biofilms, a kind of 'reef' of bacteria. They use this molecule to cut themselves loose. When we flood the area with DspB, the bacteria will not be able to make the biofilm, because they will be cut loose as soon as they try to attach themselves.

What is L. lactis (and other bacteria)?

Lactococcus lactis is a gram-positive bacteria. Gram negative bacteria have a cell wall sandwiched in between two cell membranes. Gram positive bacteria have a thick cell wall around their membrane (Figure). Lactococcus lactis is usually shortened to L. lactis. This a bacterium is found in many food products such as cheese and beer and does not make us sick. Contrary to popular belief, most bacteria do not make us sick. In fact, many bacteria benefit us and some are necessary for us to live. Your gut is filled with bacteria that break down your food into molecules that your intestines can take up. Without these bacteria, you would not be able to eat anything! In return, our bodies are a safe home for these bacteria. Such an arrangement is called symbiosis, literally translated this means 'living together'.

We do not 'live together' with L. lactis in nature, but we can make L. lactis work for us in the bandage. Like L. lactis, Staphylococcus aureus (S. aureus for short) is gram positive. S. aureus produces toxins that are damaging to humans.

Figure 2: Bacteria are made of DNA and proteins encapsulated in a membrane. They use the proteins to multiply the DNA and make new bacteria. Gram negative bacteria have a thin cell wall and a second membrane. Gram positive bacteria have a very thick cell wall.

What are burn wounds?

Burn wounds happen when the skin becomes too hot or too cold. This means that if you touch something in a freezer that is -80°C, you can burn your hand just as if you would touch boiling water. Because temperatures so low that you would burn yourself are very rare in nature, they were not recognized as burns until relatively recently and we associate burns with high temperatures.
There are several degrees that you can get burnt.

First degree burns barely damage your skin, but functions of your skin start getting affected and the skin can dry out. In response the skin gets swollen and can start to itch or even become painful.

Superficial second degree burns damage the top layer of the skin, the epidermis (See figure 1). In this part of the skin, there are no blood vessels or nerves. Blood still flows through the skin, and you still feel pain in the burnt skin. Superficial second-degree burns do not damage the basal lamina. The skin regenerates from the basal lamina upward.

Deep second degree burns go down to the middle layer of the skin, the dermis (See figure 1). The dermis contains nerves and blood vessels. Deep second degree burns partially damage the basal lamina. Thus nerves, blood vessels and the regenerating part of the skin are damaged. The sense of touch is lost in the burn, the skin loses fluid through blood loss and regeneration is slowed.

Third degree burns go all the way to the subcutan tissue (See figure 1). The basal lamina is lost, and regeneration is not possible except for the edges of the wound where there still is some basal lamina.

Fourth degree burns go furter down into muscles and organs.

Figure 3: The skin has four layers. The subcutan tissue lies between the skin and muscles in your body. The basal lamina forms the basis of the skin. The dermis contains many live cells and many nerve endings, hair follicles and glands suchas sweat glands. The epidermis is made of mostly dead cells and forms a protective layer all around your body.

Sorry, nothing here yet, but check out our burn wound education!

For now, a list of our supervisors, more info will follow.

Oscar Kuipers
Jan-Willem Veening
Renske van Raaphorst
Ruud Detert Oude Weme
Bayu Jayawardhana

Sorry, nothing here yet!

Sorry, nothing here yet, but look at the bandage and biobrick notebooks!

Week 1 (21-25 july)
In silico preparation of primers for the Gibson assembly between signal sequence USP45 and modified version of Aiia. In silico production of synthetic gene ssUSP45DspB. General preparation of lab necessities.

week 2 (28 july-3 august)
designing primers for the synthetic gene.

Week 3 (6 – 8 august):
goal: obtain all the biobricks necessary for the secreting systems of P. aeruginosa and S. aureus.

E. coli was chemically transformed with 3 biobricks in order to obtain them:

BBa_I746104: P2.
BBa_K081009: LasI (for the Detection part).
BBa_C0060: aiiA.
After which 1, 10, 20, 50% was inoculated on Chloramphenicol LB-agar plates.

The RBS, Double Terminators, and the promotor LAS biobricks were already transformed and isolated.

The clones were inoculated in liquid media were they would be prepared for miniprep.

The O/N culture was miniprepped and checked on gel, giving a positive result.

week 4 ( 11 – 17 august):
goal: assemble the promotors with RBS and gfp (BBa_E0040) with double terminator.

Biobricks pLAS, P2, gfp, RBS and Double terminator were assembled accordingly to their place in the construct with 3A assembly, ligated, chemically transformed and inoculated on kanamycine agar plates.

The O/N grown colorless clones were picked and grown on their own individual plate. Afterwards, colony PCR on all the colonies showed a positive result for assembled: P2+RBS, pLAS+RBS, GFP+Dterm.

After the colony PCR, another one was done with phusion DNA polymerase, so that the product could be used for a second assembly with (P2+RBS)+(GFP+Dterm) and (pLAS+RBS)+(GFP+Dterm) corresponding in only a few clones which gave negative results after colony PCR.
Therefore, another assembly with the constructs should be done.

Primers came in and were prepared accordingly.

The synthetic gene ssUSP45dspB, which arrived from IDT, was prepared according to the protocol made by IDT. 1 µl of pSB1C3 was cut with EcoRI & PstI. The digestion mix ran on a agarose gel, afterwhich gel purification of the linearized backbone occurred. 70ng of vector was used to ligate ssUSP45dspB.

The yield of transformants was very low after transforming the ligation mixture and growing the bacteria. Only one colony contained the ssUSP45dspB according to gel.

Week 5 (25 – 29 august):
goal: obtaining a construct with the promotors and GFP for promotor analysis in L. lactis.

All the clones containing the constructs with p2+rbs, pLAS+rbs and gfp+Dtermwere grown again to be miniprepped. After this, these products, including ssUSP45dspB ran on a gel showing that p2+rbs and pLAS+rbs had the correct size, but ssUSP45dspB and gfp+Dterm didn’t had the correct size.

Again, the gblock of ssUSP45dspB was cloned with the digestion mix, and again rendering negative results.

After a couple of failures with the small amount of gblocks which has been given, PCR was done on 1µl of the digestion mixture. The products which were produced were: ssUSP45 and ssUSP45dspB without his-tag.

week 6: (1 – 5 september)
goal: obtain ssUSP45dspB in pSB1C3 biobrick and ssUSP45aiiA in pSB1C3 biobrick.

Because of the very low amount of gblocks given, a decision was made to do a PCR directly on the product itself, therefor multiplying it exponentially.
2 series of PCR ran:

1. Amplification of gblock in 50µl reaction.
2. amplification of product: 10*50 µl reaction.
The products were ligated into 70ng of pSB1C3 and transformed into E. coli . Another PCR was done with the amplificated PCR product of ssUSPdspB, these products were:

1. the signal sequence of USP45
2. the gblock without HIS-tag.
These products were also ligated into pSB1C3 and transformed into E. coli.

20 nucleotide overhangs were created on aiiA, making it ready for Gibson assembly
Afterwards, Gibson assembly had been done with modified aiiA and the signal sequence of USP45 making ssUSP45aiiA. to enhance the chances of successfully ligating the Gibson product into pSB1C3, PCR was done on the final gibson product.
The PCR product was checked on gel, giving positive results for presence of ssUSP45aiiA, then it got ligated into pSB1C3 and transformed Into E. coli as well.
Results of the transformed E. coli gave a yield of 40% of possible clones.
Colony PCR was done on them with the regular pSB1C3 test primers, but no product was seen. Therefor 40 possible clones were grown O/N and miniprepped the next week.

Week 7 (8 – 12 september)
Goal: analysis of the possible made Biobricks.

All the O/N cultures were miniprepped. The possible plasmids were cut with EcoRI and SpeI. After running on gel, the result showed us that 20 of the 40 clones contained all the biobricks, though with a low yield of plasmids.

A new strategy of assembling has been prepared and primers for this strategy have been made.

All the Biobricks were send for sequencing.

Week 1 (2-4 July)
Two strains of L. lactis (NZ9800 & NZ9700) were freeze dried and kept in -80 to see if they could be revived the next week.

Week 2 (7-11 July)
Growth of the two L. lactis strains was tested by resuspending them in medium and letting them grow overnight. They grew very fast. The experiment was repeated with a lower starting concentration, and they still grew very fast.

Week 3 (14-18 July)
(Nothing)

Week 4 (21-25 July)
A culture of L. lactis was made to put in polyacrylamide gels.
Polyacrylamide gels were made at 15, 20, 25 and 30% polyacrylamide with L. lactis in it to see if the bacteria grow in the gel. They grew best in 20% polyacrylamide, thus a new gel with bacteria in it was made. A quarter was freeze dried immediately, another quarter was incubated for 45 minutes at 37 degrees, ther other two quarters were incubated overnight.
Cells were found in all quarters.

Image 1: One quarter of PAA gel before freezedrying

Image 2: One quarter of PAA gel after freeze drying

Week 5 (28 July-1 August)
A stock solution was made of pNZ8048G, a strain containing GFP. It was tested by activating the GFP with ZnSO4.

Week 6 (4-8 August)
To increase survival of L. lactis, a new gel was made on ice. The gel did not solidify. Another gel was made with the GFP strain inside, but the GFP did not show when activated with ZnSO4.

Week 1 (2-4 July)
An overview of necessities was made.

Week 2 (7-11 July)
Primers were designed for the Nisin operon and superfolded GFP.

Week 3 (14-18 July)
The plasmid pSB1C3 containing the CP promotor was isolated.

Week 4 (21-25 July)
Gibson assembly primers were designed for the removal of illegal sites from biobricked genes.

Week 5 (28 July-1 August)
The insertsize of the pSB1C3 plasmid was verified.
The genes PNisA, NisA and sfGFP(Bs) were successfully amplified according to their product size.
PNisI, NisA and sfGFP(Bs) were purified. PNisI, NisA, sfGFP(Bs) and pSB1C3 were then restricted to create overhangs.

Week 6 (4-8 August)
A PCR was performed with a longer elongation time and turned into a ‘touch up’ PCR, in which the first 20 steps let the annealing temperature rise from 40 to 60 °C and the PCR ends with 20 cycles with an annealing temperature of 65 °C. Also two further series of PCRs was performed, one with GC buffer and one with GC buffer and 1.5% DMSO. Now all the genes got amplified.

Week 7 (11-15 August)
The PCR of NisRK was repeated twice, the first failed because of an overload of template DNA. The diluted version did work.

Sorry, nothing here yet, but look at the bandage and biobrick notebooks!

Sorry, nothing here yet!

An early sketch for a logo and the first name/combo idea:

The first appearance of our distinctive phoenix:

Sorry, nothing here yet!

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 finale in Boston, he will fly back to Amsterdam, take the train to Berlin, and tell a large international
 audience about our bandage on November 8./p><br>
-<img src="https://static.igem.org/mediawiki/2014/9/95/FallingwallsII.jpg">
+<img src="https://static.igem.org/mediawiki/2014/d/d9/FallingwallsII.jpg">
 </span>
    <span id="content_policyandpractice_sciencelinx" class="visibilitytoggle content_policyandpractice_sciencelinx"><p>Sorry, nothing here yet, but check out our discovery festival piece!</p></span>

Team:Groningen

From 2014.igem.org

Revision as of 13:18, 2 October 2014

Home

Abstract/English

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Policy and practice/Overview

Policy and practice/Future Perspectives

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Policy and practice/Martinihospital

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Policy and practice/Events/Falling walls

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