World Championship Jamboree/blog4

Last session of the day and of the iGEM Championship Jamboree this year: track new applications.

Before the presentation starts they formed a motivation team huddle. So, Go Uppsala!

The team wants to create bacteria, which will be produce a color output (chromoproteins, fluorescence protein), triggered with a light signal = a bacterial photo copier. So the gene expression will be controlled by the use of light. Further it is possible to exchange the color-protein-genes with other GOI, which will be triggered by light. The swedish team is inspired by Jeff Tabor and his collogues, who published a multi chromatic control system for gene expression. They presented their ideas in really nice overview slides/ assembly plans. Check these on their wiki.

They implemented different light sensors (red and blue light) into E.coli. The blue light sensor works as follows. In darkness he triggers the expression of a protein. This expression is suppressed under blue light. The team coupled the sensor signal with colored proteins (blue ight => blue color, green light => yellow color, red light => red color). On the basis of these three colors an output of a lot of different colors is possible. Further they worked with a so called lambda red recombination plan, in order to get more stability (chromosome based), more control and to avoid crosstalk. So, how does this work? First of all you amplify your sequence by PCR. Here you create new flanking tags off your DNA part. These flanking parts should be homologue to the sequence inside the chromosome. The construct gets transformed into your bacterial chassis and afterwards you do a selection.

They talk about their favorite parts. Looks pretty colorful. And sure they characterize their parts as well as their used promoters. And sure there is a human practice part. Visiting schools, being in the newspaper and in the radio. Nice work guys.

Somebody fell asleep a few rows in front of me and woke up snoring loudly. Shame on you dude. Get yourself together. This project is definitely worth being awake.

Visit their wiki!

To develop flexible and better sensors for environmental, agricultural and engineering applications are the aims of the UTP-Panama Team “SynBio Engineering Tool Kit”. In this way we work with Nitrate Biosensor (PyeaR – GFP composite) developed by Team BCCS-Bristol 2010, which expresses fluorescent signals upon nutrient detection, producing a high-resolution map of arable land. To achieve this goal we use the collateral effect of the AOX enzyme (Alternative oxidase) mainly designed to generate heat in response to a cold-shock, using the hybB promoter. This effect increases the bacteria growth at temperatures below 20°C. Finally we design a prototype device with a better cold shock promoter (CspA promoter) developed by UNAM-CINVESTAV Team in 2010, in order to give our E. coli a “Intelligent Coat”, which means that not to only survive a cold-shock but to also still been able to keep up with his duties due to improve their expression mechanism at low temperature.

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The UTP-Panama team seeks to create more robust sensors that function at low temperatures. They chose the Nitrate Biosensor (PyeaR – composite GFP developed by BCCS-Bristol 2010) that has agricultural applications—if this sensor can be made to work at lower temperatures (than 37oC), it will have be more agriculturally relevant!

Their THErmogenic REsponse Nutrient BiOsensor (THE RENBO) construct puts this biosensor under control of a cold shock-sensitive promoter. The team used this construct to assess the activity of the biosensor under different cold shock conditions and found that it could sense nitrate at lower temperatures…

For their human practices, UTP-Panama looked at the diversity of human practices projects across all iGEM teams and developed an outreach campaign that educates and then examines their target audience. Exam results were on average a B grade! Please find their software on their wiki HERE. They have also contributed significantly to CommunityBricks in hopes of spreading the word about synthetic biology all over the world!

The team that accidentally started crazy music while the ITESM Mexico was presenting. Face palm.

An introduction concerning the earthquake that led to the Fukushima nuclear crisis, the most severe nuclear crisis in Japan. Negative biological effects of radiation are shown. A human practices survey is shown where most the people associate negative terms with “radiation”. Seems like no huge surprise to me.

Main idea of the team is to design a bio-dosimeter which has some advantages to common dosimeters. Two main aspects of the design plan are presented: Damage tolerance and detection of DNA damage. Since Deniococcus radiodurans has a very high resistance towards radiation and a very effective DNA repair mechanism, it shall be used. The team cloned various DNA repair genes from Deniococcus radiodurans. Afterwards, different tolerance assays were performed. They could show that the IPTG induced expressed of these genes led to an increased tolerance/viability. When combing the parts PprM and RecA, they showed the effects are additive.

Thereafter, the DNA damage detection, which is focused on the OS response, is shown. A damage detection device using the SOS promoter and a reporter gene lycopene is used. The cells are damaged with UV radiation and the OD measured. They could show that the SOS promoter could be induced by UV radiation.

For the future, the team has a lot of promising plans.

Visit their wiki!

Antifreeze proteins have applications in cryopreservation of food, cells, and organs, as well as in cryosurgery and agriculture. The purpose of this study was to express, purify, characterize, and optimize a novel, hyperactive antifreeze protein recently isolated from the Siberian beetle, Rhagium inquisitor (RiAFP). Large scale (150mg/L), stable production of RiAFP and a RiAFP-GFP fusion protein was achieved in E. coli. Proteins were purified by Ni-NTA affinity chromatography. E. coli expressing RiAFP exhibited increased survival post-freezing. RiAFP inhibited ice recrystallization in a dose-dependent manner. RiAFP also improved tissue morphology of rat livers post-freezing. Preliminary results indicate that RiAFP may have a cryoprotective effect in C. elegans. To optimize the activity of the hypothesized RiAFP binding site, we used directed evolution through multiplex automated genome engineering and are currently screening for mutants with enhanced properties. Finally, to better understand the structure-function relationship, we have generated promising crystals of RiAFP for x-ray crystallography and are now optimizing crystallization conditions.

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On a cold february day in New Haven, the Yale iGEM team decided to study antifreeze proteins (AFPs)…

AFPs can be found in organisms dwelling in cold environments such as insects and fish. AFPs are conventionally characterised by how they affect the melting point of water… but more interestingly, AFPs are used in various industries, particularly in ice cream production!!

Ice cream today uses fish AFPs which are much less effective than insect AFPs that are limited to laboratory use. The Yale team looks at the RiAFP, a novel hyperactive AFP from the Rhagium inquisitor beetle. They have successfully characterized this as well as 2 other AFP biobricks through measurement of expression of the eGFP-AFP fusion proteins.

These AFPs were purified via cold finger; sticking a cold metal tube seeded within a thin layer of ice that AFPs bind (their natural property). This is comparatively cheaper than conventional purification methods and is specific to AFPs.

With Yale’s infrastructure for structural characterization, the team obtained X-ray crystallography diffraction patterns of their RiAFP.

They demonstrated RiAFP’s ability to improve freezing tolerance in different organisms: E. coli (bacteria) and rat liver tissue (rat work performed by licensed personnel).

To take their project to another level, the Yale team embarked on multiplex automated genome engineering (MAGE) optimization of AFP activity. To date, they have generated four hundred and thirty four million predicted genomic variants that will be tested via multiple freeze-thaw cycles.

Second session, first talk, environmental track

The speakers suited up and they got a very nice mascot – the biobrickcoatl! The mexican team shares a dream, bringing technological ideas into biology – taaadaaa iGEM! So they applied for the competition. After refusing different ideas, they want to work with a biosensor. Hence, they reviewed different projects from other iGEM Team (Monterrey iGEM, Cambridge, Tokyo etc..). Finally they called their project SensE.coli. SensE.coli The project consist of a standardized Biosensor, which can be used in different applications. They chose to build an arabinose biosensor. The project can be divided into two big sub-projects; A photoreceptor, which is activated by the receptor and the promoter,which produces the RecA protein.

Under green light and low arabinose concentration conditions, the low conc. promoter is induced. This leads to the production of the key for the GFP production and GFP is produced. In high concentration conditions, the anti-key for the GFP-key is produced and suppresses GFP-production. Hence CFP is produced. So the Biosensor is able to differentiate between low and high concentrations of arabinose. The user gets a different color-output for different concentrations. I am wondering at which concentration the switch might be and how sensitive this kind of biosensor is…

The first, which pointed out that iGEM is fun and fun should be in the results part. You are damn right! Concerning the wetlab work, they were not able to test their whole system. So they broke it down and created a express test system. They manage to find some of their DNA via gelelectrophoreses, but they pointed out that the PCR primers, necessary to proof the principle of the mechanism, are still under construction at the DNA synthase factory (it seems that it takes about 4-5 weeks in mexico. unbelievable.)

The judges are concerned about the strict regulation in mexico, which seems to block the success of the team (sequencing and ordering of parts). It seems that every mexican team deals with this kind of problem. The judges suggest to do some human practice projects on this problems. So all in all, these guys did a good job concerning their struggling with the mexican government.

Visit their wiki!

We have developed a pipeline for selection and characterization of new transcription factors (TFs), specifically:

1. in vivo selection offunctional mutants from a large library of variants using a “survival of the weakest” strategy;
2. in vitro characterization of affinity and specificity of mutants with MITOMI;
3. in vivo characterization of selected mutants using reporter plasmids.

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Their protocol selects for TFs (their teenage mutant ninja turtles) with high binding affinity by

1. in vivo by lysis selection

- Using lysis cassette (Berkeley 08) to lyse cells with effective TFs and release TF-encoding DNA

- They controlled for lysis rates by comparing the amount of plasmid in supernatant in their desired strain versus control

2. in vitro by microfluidics (in vitro findings were later confirmed in vivo)

- Measure TF-DNA interaction over 5 hours

- Focus on TetR repressor mutants and their binding affinity to the promoter using RFP reporter

For teams interested in incorporating microfluidics into their project, the EPF-Lausanne team (which built their own accessible microfluidics setup) recommends ordering your chip from existing foundries to avoid dressing up like an astronaut and making your own ;)

1.7 million hospital acquired infections, from that 170000 caused by pseudomonas aeruginosa. The team from Northwestern University wants to create an detector (E. coli biosensor) for that bacteria, using quorum sensing signals from P. aeruginosa to induce their reporter.

Their detection system based on LasR is explained and tested. You clearly see the effect of the autoinducer in comparison to the negative control. Unfortunately, there is no distinct differentiation between different concentrations of the inducer. Or in other words: It’s a binary outcome.

Rhl Based Detection System, now.
This time, the output fluorescence is clearly dependent of the autoinducer concentration. They show two different versions of this system – both seem to work pretty well.

They could nicely model their system, showing predicted GFP concentrations over time for different inducing concentrations of LasR or RhlR respectively. Their model builds upon hill equations. The critical parameters for that model are the degradation rate of the R protein and the degradation of the dimer of LasR and PIA-1, RhlR PIA-2 respectively.

They imply that they can optimize their construct to “tune” its sensitivity.

Applications: We see pictures of a possible stick to use for P. aeruginosa sample pick-up and a LED-box to detect the resulting fluorescence.

The team submitted 38 parts, 19 of them fully characterized.

A content-packed presentation finds it end. Find more information on what they did on their wiki: https://2011.igem.org/Team:Northwestern

Second Day, first session, second talk, track medicine

The brazilian team took some kind of a yoda coli along: Very nice artwork. I am excited. The presentation is designed in the star wars look. Nice.

The team wants to deal with pathogenic stress, which everybody suffers from time to time or constantly. Like me, blogging all the day :) They describe the interplay between the naive t-lymphocytes and the t-helper class 1 (Th1) or 2 (Th2). In a pressure situation, stress hormones are released, which lead to a shift in th2 (th1 suppression) and low cellular immune response. The low immune response can promote an infection caused by microorganisms. They want to create the JEDI coli that can sense stress and drive the naive t-cell differentiation.

They use oxidative stress system and a quorum sensing system to sense the immune imbalance (NO-sensing) in stress situations. The application of the project might be the modulation of vaccine response or treatment of diseases caused by immune in-balance. They introduce two different systems, which in stress situations battle against each other. This is done by the production of different molecules (cathelolamines), which can trigger the th1 or th2 concentration respectivly (Interleukin 12 and 10). The two systems interact which each other. This is done in the following way. If one systems gets triggered by a stress symptome it will start the production of its interleukin, in order to trigger th1 or th2. This mechanism works vice versa. The brazilian team characterized their producing systems with gfp.Unfortunately their system is a little bit too complex to explain it here in detail respectively to blog and hear it at the same time. Or I am just too tired. Check their wiki for full information. It is definitely worth it. C’mon they got YODA!:

https://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project

Morning session, first talk. For 9 AM the room is quite crowded. Let’s see what the local team has done the last months.

Programmed Mammalian Tissue Engineering - Local interactions to global patterns

A short introduction to their main topic: The need for tissues which is a global problem since the current approach is solely based on human donors (18 deaths per day waiting for organs). The team wants to use the synthetic biology approach for tissue engineering and has focused on pattern formation. They present their system design where two phenotypes are possible – a sender and a receiver phenotype – and where three main modules are implemented. These modules are: cell-cell signaling, internal logic processing, and cadherin output.

A nice 3D animation showing how the final system should look like, is presented. The first component of their genetic circuit is a Notch-Gal4 which finally leads to the expression of cadherin and Delta-mCherry. The presented system is tunable with IPTG. Through cis-inhibition the expressed Delta-mCherry can shut down its own notch or activate other cells.

They also used a computational design tool to see what other interesting genetic circuits can be created. A simulation is shown how a group of cells forms a pattern after 24 h. They used their tools to simulate other designs and parameters.

Now to the experimental results. They started characterizing their parts one by one starting with UAS-Gal4. Afterwards the lacI repressor is tested. Gal4 connected to an inverter and a tunable amplifier are checked. Finally all the parts are put together (Inverter/Amplifier/Sender) and the mCherry fluorescence is measured. They can show that both, the sender and the receiver module works. This is underlined by some nice confocal microscopy pictures. It’s always cool to have something visualized. ;) Nice presentation.

Last talk in this session, last talk for the track information processing and the last talk for today.

The complete team is at the front. The topic is the construction of a molecular AND gate in E. coli that may be used for example in the detection of early onset of colorectal cancer. Since two features, heat and reactive-oxygen species are the two hallmarks of colon cancer, an AND gate will be a great way to detect these cells.

A nice stop-motion video is presented.

Now, modeling. The team used various differential equations and shows some nice figures, but it is not possible to describe these figures in words…you have to be a robot/transformer.

A thermosensor from Listeria monocytogenes that is used for infection will be used in the project. The problem is that the team needs a thermosensor with a narrow temperature range, but details concerning that will follow later.

They used the thermosenor for proof of concept experiments. A pSox promoter is also used and proofs that the AND gate is functional and that parts are interchangeable.

They tried to rationally design a thermosensor with a narrower temperature range. Also random mutagenesis was tested. A huge screening was performed (“100s of thousands”). The team was able to find novel 30-37° C thermosensors and also thermosensors with a really narrow temp range…35-37 °C. Congrats. After designing the new thermosensors, they started characterizing them so that they could go back and do a rational design again. For this, they started with some folding software.

Community outreach. DNA extraction from strawberries at a local elementary school. “Is that really DNA? – Yeah.”