World Championship Jamboree/blog5

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Cornell’s 2011 iGEM team has designed a new, scalable, and cell-free method to produce complex biomolecules. Current methods for purification from cellular lysate are expensive and time consuming. BioFactory utilizes modified enzymes, capable of being attached to surfaces, in the creation of a modular microfluidic chip for each enzyme. The surface bonding is performed by the well-characterized biotin-avidin mechanism. When combined in series, these chips operate as a linear biochemical pathway for continuous flow reactions. Additionally, we plan to engineer E. coli with the mechanism for light-induced apoptosis to easily lyse cultures producing the necessary enzymes. The resulting lysate is flowed through the microfluidic channels, coating them with the desired enzyme. We believe this chemical synthesis method will reduce unwanted side reactions and lower the costs of producing bio-pharmaceuticals in the future.

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Nice opening animation; cue dramatic music. They are SUPER excited to be here today.

Cornell’s project involves designing, building and testing a new method of manufacturing biochemicals outside of cells. The problem with biochemical synthesis in cells is that there’s a ton of problems involved, including hard-to-control conditions (pH), toxic or unusable side products and sometimes long purification steps.

Isolate enzymes and use a microfluidic device to optimize a compelx reaction. For a POC they looked at a 3-enzyme pathway involving L-tryptophan. They wanted a high surface area, modular and scalable microfluidic system with enzyme-studded channels. To get the enzymes first they lyse the cells, which could be costly with huge bioreactor-sized cultures. So they looked at light-inducible lysis.

They get enzymes to stick with the avidin-biotin reaction, tagging their enzymes with an “AviTag”. They found no negligible effects of flowing cell lysate through their channel. Enzymes will get knocked off from the fluidic flow, so they need to be refreshed every 15 hrs.

Cute animation. Future application – using a large pipe with enzyme-coated beads, kind of like Caltech’s biofilm for endocrine remediation.

Human Practices: science experiments with kids, teaching high school girls molecular biology, Community Bricks.

Last talk in this session.

The team deals with diesel production and gluten destruction. Two very similar topics. First they focused on the diesel production. They want to use Biofuels, because these are renewable sources of energy and mix them with petroleum to get the right mixture of good fuel. The alkane production should be managed by using fatty acid as a starter substrate. This pathway was implemented , expressed it in E.coli , analyzed it via GC/MC and calculated a model. They achieved a production of C13, C14,C15 and C17 Alkanes and Alkenes respectively. So they can convert sugar into diesel, as they expected! They called it the PetroBrick, which is able to produce alkanes of different chain length.

The second topic is the gluten degradation. Some people suffer under gluten side effects. The only treatment is, a total dietary change. Gluten peptides trigger a immune response. So they want to digest these peptides. There is a protein already in test trail, unfortunately this protein is not able to work at a low pH. So they choose kumamolisin, which can work under low pH conditions. They used computational tools to redesign kumamolisin for their purpose. The team developed a whole cell lysate assay, in order to test their mutations. The top mutants were purified and characterized. These could be increase to a 100-fold improvement of breaking down these bad gluten peptides.

Further the Washington Team achieved to create a iGEM toolkit box, which can be used by the iGEM community. I like toolkits. Nice work.

I do not really understand, why they choose three totally different topics. However, I have to say that they did a really nice work on all of their subprojects.

Just a quick side comment. The last girl can speak with the speed of light or like a automatic gun. 1000 words per seconds. Literally. And counting. I am blown away.

Q&A

Why do you choose such different topics?

Because they were soooo many people, they did different topics. The connection between the topics seems to be making the world a better place with the help of synthetic biology.

What about your application concerning the biofuels

Just a proof of principle. There is a need for more work in order to get the best yields.

A biorefinery is a special type of refinery in which biomass, such as plant cellulose, is converted by microorganisms into useful products. Edinburgh’s 2011 iGEM project is a feasibility study into the creation of biorefineries using E. coli, the workhorse of synthetic biology; and whether biorefineries can be improved by arranging for the different enzymes involved to be in close proximity to each other, so as to create synergy between them. We investigated two methods of bringing the enzymes close together: cell surface display via Ice Nucleation Protein, and phage display via M13′s major coat protein. We attempted a new DNA assembly protocol, provisionally named “BioSandwich”. We constructed computer models of synergy to assess whether it is a feasible option. Finally, we considered the broader economic and social questions surrounding the construction of a biorefinery: can it be done, and should it be done?

The team presents the concept of synergy – within their team and within nature. Afterwards, they transfer this concept to two different strategies – cell surface display and phage display.

The first presented system uses E. coli as a scaffold and displays proteins on its outer membrane. To achieve high expression levels, they attempted to use Ice Nucleation Protein as a carrier for enzymes. They show promising results where they could degrade cellulose and also starch. Nearly not necessary to mention, but the team did some modeling and even though they say it’s quite simple, they have lost me. They also compare stochastic and deterministic models.

A new protocol named BioSandwich, a method for the assembly of multiple BioBricks in a single reaction is presented. The protocol seems pretty simple and consists of only five steps. You can find a lot of details on this in their wiki.

They performed a feasibility study. I suppose this is the only team that has been doing such an extensive study to see if their project is applicable and how one could realize it. Furthermore, they have performed interviews with various persons from politics, academics and even a member of the Church of Scotland. This is probably the most outstanding part of the Edinburgh teams project. I suppose very few iGEM teams before have been covering so many outreach and human practices aspects. Really impressive.

Third session. First talk of the Food or Energy track

Before the talk starts, somebody has some really serious problems with the light switch. It’s on, off, on, off again. Gives me the disco feeling.

So, back to serious topics. The team is dealing with the big problem of worlds hunger and malnutrition! They pointed out that is not only a problem of quantity, but also quality. You need your vitamins! Infant and maternal care as well as sustainability are the two other main issues. The JHU team focused on vitamin A and C. The project consists of a theoretical, modeling, toolkit and a deployment part. As a chassis they choose yeast, because it is already evaluated in the scientific community and has been used for centuries. The main aim is to create a vitamin A and vitamin C producing Yeast. The Vitamin A should be produced from the beta-carotine pathway. They transformed the used genes in different Biobricks, transformed it into the yeast and viola you get orange yeast (do not try at home). For the Vitamin C pathway they choose GDP-D-Mannose as a precoursur to produce L-ascorbate. They transformed all the used genes into BioBricks. For purification a his-tag was used and enzyme assays performed. And of course, there is a model. Unfortunately, it is math, so I am out. Sorry guys! But it seems pretty nice. They calculated a Pareto Frontier, which seems to be the calculation of the turning point of the system. At which time point do I have to supply to much nitrogen, without getting more product. Interesting and well performed project. But the questions is, what do they think about golden rice, which solves the same problem like their Vita-Yeast.

Further they supply a yeast toolkit, containing promoters, UTRs vectors and reporters. Toolkits are always a great idea, because they can really help other teams out. Nice work!

Finally they liked to baked bread by using their optimized yeast. So people can get more vitamins by eating their bread. The prototype bread is not orange, but looks like real bread! They measured their beta-carotine amount by HPLC. So there is beta-carotine in their bread. With this idea they went out of the laboratory for their Human Practice part. So finally they mantioned the golden rice, and claimed that their bread will be better, because it is not orange. Further they say, because their bread only contains 1% of GMOs, instead of the golden rice, which is a complete GMO itself….

Let’s check the Q&A part.

What about the instability of your vitamins?

They did consider that the vitamins can be degraded under heat. So they want to produce more than required and they claime that the compounts are stable under baking conditions

2 questions I did not get not acoustically.

Last presentation before lunch break – The cobalt busters from Lyon. I hope no growling stomachs will interrupt the presentation. ;)

How to remove cobalt from radioactive water?

Another tribute to one of my favorite childhood series. Humming the title track, I keep following the presentation.

They demonstrate how radioactive cobalt is formed in French nuclear power plants, originating from the steel pipes. The cobalt is part of the radioactive waste which has to be stored. Since space for storage is expansive and a problematic issue, the team has come up with a new way to threat with the waste.

As already heard in the Grinnell presentation earlier this morning, biofilms are involved. But surprisingly, this time the advantageous aspects and the potential of biofilms in bioremediation are pointed out. Since they want to use a modified E. coli chassis which is accumulating cobalt, a separation of the bacteria and the waste is beneficial and for this they want to use biofilm formation. Two different options are presented: A synthetic culi operon and by activation of cryptic curli genes. Results for the characterization of both ways are presented. They can show that their device employing the first option works as desired and displays increasing adherence with increasing cobalt concentrations. Also, they show that their rcn promotor is the reason for that behavior. After comparing both possible ways, the first option is selected.

Now, for the actual implementation of their bacteria. The team presents a figure how a filter carrying their bacteria would be used in the bioremediation of contaminated water. Also, further applications such as air filters are presented. Like nearly all of the iGEM teams, they have been heavily working on the iGEM outreach.

I hope that you all are as hungry as your designated bloggers are.

Find the mercury in water! Mercury is a problem for water-quality all over the world. That’s why the Grenoble team started a partnership with an industrial partner from chemistry to establish a detection and quantitation device for mercury, based on our friend E.coli.

They use a plate with constant mercury concentration and an IPTG gradient to start expression in either sending (mercury > IPTG) or receiving (IPTG > mercury) bacteria. For that, they created a 2-way switch, which allows to distinguish between higher Hg or IPTG concentrations and will cause quorum sensing from receiving bacteria to sending bacteria. As a result, the receiving bacteria will accumulate at the equilibrium of Hg and IPTG and create a coloration, there. That again, allows to read the mercury concentration according to the IPTG concentration gradient. They additionally implemented a power button for the whole system. See their wiki for a way better explanation and helpful images :)

They show some models, to make clear how they expect the results to look like. They modeled predictive outcomes.

As Grenoble is a pretty interdisciplinary team they aim to improve the interaction between biologists and modelers. For that they created two info flyers “Modeling for Biologists” and “Biology for Modelers”. They tested their flyers in a collaboration with Paris, where they had two test groups – one using the flyers, the others not – and a quiz for evaluation. The results proof the usefulness of their attempt. And finally somebody can explain modeling to me. Nice!

In the human practice part, they show some actions in science communication, including conferences, newspapers and radio.

All in all they could make 3 proofs of principle (2-way switch, power button and quorum sensing), committed 19 BioBricks and MOST IMPORTANT they will be back in 2012 with the next Grenoble iGEM team :)

Time for questions:

Didn’t understand the question..
They showed, that their systems works for Hg and tetracycline.

Couldn’t understand the question…
But the answer is, that you need 10^4 bacteria to achieve a 10% precision in the system.

Please repeat the questions :) …
For creating the information flyers for modeling and biology, they switched the roles in the team, to make sure to hit the important questions that arise, when a biologist tries modeling and vice versa. Their goal was to bring the basic knowledge of both worlds to every team member in order to allow an efficient team work.

What is the response time of the system?
It is about 1 hour.

Does their system work for other substrates?
It should be easy to use other sensors for the same basic concept.

Are their requirements (37 degree, 1 hour response time) realistic for real world applications?
They seem to use only bacteria in exponential growth for their test, making it independent of such limitations.

“Check one, two” and the presentation starts.

The iGEM Bielefeld-Germany team deals with a cellfree biosensor this year, used to detect the possible toxic and polluting substance bisphenol A (BPA). Reminds me of my old 80ties heroes.

BPA is used polycarbonate plastics, hence we get in contact with this substance every day (DVD,CD, baby bottles). There are concerns that BPA, because of mimicing estrogen, is bad for your health. Hence it is forbidden in the EU and Canada for the production of baby bottles (Never let your baby suck on a number 7).

They checked for advise by the project manager of synthetic biology of the federal agency of technology assessment. The project manager adivised: Go cellfree, where ever it is possible. In a small animated video the team explained their project. They fused three, BPA degrading, enzymes to a silica bead by using S-layer proteins. The visual light signal was established by detecting the NAD+, which is created during the enzymatic BPA degradation steps, with the help of a molecular beacon. So the detected amount of NAD+ correlates with the degraded BPA. SO the project can be divided into three subparts : BPA degradation, NAD+ detection and S-Layer.

They showed that the BPA degradation works fine by creating a double and a triple fusion protein out of the three enzymes. Further the NAD+ detection works as expected and can be checked by the naked eye. This kind of detection can be used by every future iGEM teams => nice.

The S-layer proteins were used to couple the enzyme reaction onto the silica beads. S-layers proteins self-assemble in solution or on surfaces. Further they have the big advantage that the coupled enzymes are ordered in a defined orientation. They created a DIY-Nanotech Guide, so that every other team can use this guide to build up their own S-layer based systems. Seems as if it works fine.

The Human Practice part consists of high school visiting, debating their project with the industry and public discussion. So it seems that they love it when a plan comes together and I will now check for my cigar.

Questions:

The judges like the idea of going cell free, but they are wondering about the NAD+ detection. Might there be any interference with this kind of bio-assay, especially by applying a complex matrix.

In the case of their study, there is no complex matrix, but if you use a complex matrix there might be interference.

Did they do the microscope image by themselves?

No, they took the photo out of their working group, not enough time to establish an atom force microscope-strategy.

Did you try any other substrate than silica beads?

Just silica beads and silica weavers. But they observe that their S-layer could stuck on the bacterial membran and the literature sugest that they might stick to gold particles

Why use your biosensor and not just ban the BPA?

They are scientists, no politicians.

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Endocrine-disrupting chemicals are substances which detrimentally effect the development and reproduction of wild organisms. These chemicals mimic natural biological estrogen in their interaction with animal estrogen receptors. This interaction has negative effects for reproductive processes of several species of fish and birds. To remedy that, the Caltech iGEM team hopes to engineer bacteria which can degrade DDT, synthetic estrogen (17a-ethynylestradiol), bisphenol A, and nonylphenol to less toxic forms. Compared to traditional forms of pollution removal, bioremediation is relatively cheaper and less disruptive to the environment. However, a successful project must make sure that the bacteria used for remediation do not act as pollutants or introduce toxic byproducts into the environment.

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Investigated BPA, DDT, Estradiol, Nonylphenol. Proposed system passes contaminated water through purification column with biofilm- coated beads.

Looking for a degradation pathway:

1. Gene Fishing – looked for microorganisms in the LA river. As it is heavily polluted, organisms which can metabolize compounds of interest might already exist. Grew water samples up with minimal media and one of 4 compounds to screen for relevant microorganisms.
2. BisdA and BisdB
3. DDT Dehydrochlorinase
4. Cytochrome p450 analysis

Feasibility Analysis:

1. Biofilm construction – verified that E.coli would grow as a biofilm on the glass beads.
2. Water plant integration analysis – looked at 3 existing water treatment plants.