The 2015 iGEM High School Winners

This team won the High School Grand Prize at the 2015 iGEM competition.

TAS team pic photo credit: iGEM foundation and Justin Knight

The team is from Taipei American School (TAS) in Taiwan. This is the link to their wiki page:

http://2015.igem.org/Team:TAS_Taipei

What was their project about? What made it a winning project?

I was curious and looked for answers on the team’s wiki page. Here is a summary of what I found out and of how I think the project unfolded.

PROJECT IDEA AND DESIGN

The project’s goal was to PREVENT TISSUE DAMAGE FROM CHRONIC INFLAMMATION. This is a complex medical problem affecting many people around the world. Available treatments vary in their effectiveness and secondary reactions. So, the problem was definitely worth tackling – a good choice!

Inflammation is our body’s response to stuff identified as abnormal or foreign. We deal with plenty of such stuff all the time. For example, we may get skin cuts, or sprained muscles. Or we may ingest or inhale nasty substances, or unfriendly microbes. To all these things our body’s first response is inflammation – a normal event which starts the repairing process. But, after a while, inflammation is supposed to go away, and sometimes that doesn’t happen. And inflammation which persists – chronic inflammation –  is abnormal and creates troubles. This website explains.

Why you should pay attention to chronic inflammation

How did the team tackle the problem?

They probably started by reading the scientific literature on chronic inflammatory conditions, which is how they learned of an enzyme called GzmB, short for GranzymeB. If you’re not too sure what an enzyme is, here is a helpful website.

What are enzymes?

Now, back to the project… This particular enzyme (GzmB) is made by cells of the immune system. Its purpose is to help in clearing infections and tumors.  The link below contains specific information about GzmB and its family of enzymes (called Granzymes). I hope you can get the general idea despite all the technical terms.

Role of Granzymes

In short, GzmB production increases in inflammation and, when inflammation persists, GzmB persists too. It functions by getting sick cells to commit suicide, which is good. The problem is, GzmB affects not just the sick cells but also their surroundings. It destroys healthy proteins from the space in between the cells (the extracellular space). This results in tissue damage and has serious health consequences especially in the elderly.

To prevent this tissue damage, the TAS team decided to create a bandage and skin cream, which would inhibit the extracellular GzmB at sites with chronic inflammation. The bandage (and cream) had to deliver an inhibiting substance; something that would bind and inactivate the extracellular GzmB.

What could they use?

Looking through the scientific literature, the team found a study about a human protein called AntiChymoTrypsin or ACT. This protein resides in the extracellular space, and the study showed it can be mutated in a way that makes it able to bind and inhibit GzmB. The specific mutations were “borrowed” from an inhibitor of GzmB found in mice.

So, the team decided to use the mutated ACT described in this study (ACT3m) as their inhibiting agent. And they came up with a genetic circuit designed to cause E coli bacteria to produce and secrete this inhibitor in a controlled way. Simply put, they created the DNA blueprint for a biological generator of the ACT3m. Here is the device they set out to built:

GzmB inhibitor device

image credit: 2015 TAS iGEM team

The DNA parts of the genetic circuit are:

  • Promoter to switch ON gene transcription at temperatures of 37 Celsius or higher
  • Ribosome Binding Site (RBS) to start translation of the gene
  • Coding sequence for fusion protein made up of a secretory signal and the mutated ACT; the YebF secretory piece works by tagging proteins attached to it so bacteria can secrete them
  • Double terminator (TT) to mark end of gene transcription

How did they build this genetic circuit?

WETLAB

The wiki page provides a detailed description of the assembly procedure. You’ll need to be somewhat familiar with synthetic biology and how BioBricks work in order to make sense of it. Here is a summary.

  • The ACT gene was synthesized; done by commercial DNA synthesis
  • The DNA sequence of the ACT gene was mutated to remove illegal restriction sites; done commercially by mutagenesis
  • The DNA sequence of the ACT gene was mutated resulting in ACT3m; done commercially by mutagenesis
  • The ACT3m gene was added prefix and suffix sequences for BioBricking; a prefix containing EcoRI and XbaI sites was added before the start codon, and a suffix with SpeI and PstI sites was added after the end codon; done by PCR with primers that included the required restriction sites
  • The ACT3m Biobrick was cloned upstream of the double terminator
  • The YebF signal sequence was added prefix and suffix for BioBricking; done as for ACT3m
  • The YebF part was cloned downstream from the temperature-sensitive promoter + RBS where it replaced the part for Green Fluorescent Protein (GFP)
  • Final DNA assembly: The [TempSens promoter – RBS – YebF] fragment was cloned upstream of the [ACT3m – doubleTerminator] fragment; the completed device was verified by PCR and sequence analysis
  • The functionality of the genetic circuit was tested by checking if the ACT3m protein was produced and secreted; done by SDS-PAGE and the biuret test

By the end of this procedure the team obtained engineered E. coli bacteria carrying the desired genetic circuit. Along the way, many different DNA parts were created and submitted to the iGEM Registry.

But the project didn’t end here. Remember that the team wanted to use these engineered bacteria to treat chronic inflammation.

How would the engineered bacteria help?

The team thought of two ways:

(1) PUT THE BACTERIA IN A BANDAID

One way was to grow the engineered bacteria in a lawn inside a bandaid with a semi-permeable membrane separating bacteria from skin. The bacteria would secrete the GzmB inhibitor (the ACT3m protein), which would be small enough to pass through the semi-permeable membrane. At the same time, the bacterial cells would be kept inside the bandaid and away from the skin since they would be too large to cross the membrane.

Exp_prototype

credit: 2015 TAS iGEM team

But what if some bacteria escaped?

For such a scenario, the team designed a second genetic circuit. This so called “safety circuit” would not allow any engineered bacteria to survive outside the bandaid. For this they used a gene from a virus which infects and destroys E coli: T4 Endolysin. When this gene is expressed, the bacteria die. Therefore, this killer gene must stay silent inside the bandaid and must be expressed only in bacteria that escaped outside.

Exp_safety

image credit: 2015 TAS iGEM team

How would the safety device work?

It would work by placing the killer gene under the control of a repressible promoter, and adding the repressing agent – DAP=diaminopimelic acid in this case – to the growth medium inside the bandaid. This would keep the killer gene silent inside the bandaid. But, if bacteria escaped, the absence of repressing agent would cause expression of the killer gene to switch ON, and the bacteria would get destroyed.

Has the team actually done all this?

As far as I can tell, they only did part of it:

  • They built a prototype bandaid and tested the semi-permeable membrane for its ability to keep bacteria inside, while allowing secreted proteins to pass through. But no lab tests were done with the anti-GzmB device; they only tested an analogous device carrying the Green Fluorescent Protein (GFP) instead of the anti-GzmB protein. This was because GFP functions as a reporter, which means it can be readily seen.
  • They assembled the safety device and stopped short of sequencing it.

(2) MAKE THE INHIBITOR SEPARATELY, THEN ADD IT TO SKIN CREAM

The other idea the team had was to grow the engineered bacteria in batch culture from which they would extract and purify the ACT3m inhibitor protein. This purified protein would be added to a cream, along with additives that would increase skin penetration. From research in the scientific literature, the team came up with suggestions for possible additives, such as micro-needles or transferosomes. But none of this was pursued experimentally.

 

To turn ideas into a real treatment you must do extensive research. And, often times, simulating the real thing using mathematical modelling helps you along. Modelling brings a better understanding of the process, it guides the design and improves the final product. Here is my take on the team’s modelling work.

MODELLING

What needs to happen for this treatment to work, the team asked. What kind of processes must take place? A functioning bandaid/treatment would require the following sequence of events.

  1. Turning ON the temperature-sensitive promoter
  2. Producing the inhibitor protein (ACT3m) and secreting it outside the bacteria
  3. Diffusion of the inhibitor through the semi-permeable membrane of the bandaid
  4. Transportation of the inhibitor through the skin and to the site of chronic inflammation
  5. Binding of the inhibitor to the GzmB molecules in the extracellular space

Each of these processes can be described by a mathematical function that shows which variables affect the process, and how. These mathematical functions can be used to run virtual experiments on the computer, and the results of the simulations are compared to real experimental results. From this comparison (or fit) you can gain important knowledge about the real processes. And this knowledge, in turn, helps you improve the design.

The team focused on processes #3 and #5. Here is – without much detail – what the team did:

  • They used Fick’s first law of diffusion to simulate the diffusion of the inhibitor (ACT3m) across the semi-permeable membrane (process #3). This helped them understand and predict how the rate of diffusion was affected by two variables: (1) concentration of inhibitor, (2) thickness of membrane. And, since these variables can be adjusted by the experimenters, this information is useful for improving the diffusion/design. The team called this simulation the bandage flux model. But it’s not clear to me where the diffusion data came from.

Flux model

image credit: 2015 TAS iGEM team

  • They used Hill equation to simulate the binding of two inhibitors to GzmB: the mouse inhibitor which inspired the mutations introduced in ACT3m, and ACT3m itself. The use of Hill equation is a “classic” approach to understanding interactions of biomolecules. The real binding data came from previous publications. The quantitative information gleaned from the fitting process led to the conclusion that, between the two, ACT3m was the better inhibitor. And this was presented as good news since the ACT3m was the inhibitor their device was designed to produce and secrete.
  • They used the binding constants and coefficients from Hill equation to create a calculator which would tell how much ACT3m inhibitor was needed to bring GzmB levels back to normal in any individual patient. This could potentially help in customizing the amount of inhibitor in the bandaid to the needs of individual patients.

 

How realistic was the treatment proposed by this project? How safe? What were its chances to make a real difference in the life of patients with chronic inflammatory conditions? How did it affect the society and the environment? The Policy & Practices section of the project describes how the team answered such questions. I provide a brief summary below.

POLICY & PRACTICES

Like any iGEM project, this too aimed to make an impact on the real world. Of course the implications went far beyond the lab and school. Many different aspects had to be considered, such as possible side effects from the treatment, or how it would be received by patients unfamiliar with synthetic biology. And so, expert opinions and information had to be gathered, and ideas were shared and discussed extensively. Ultimately, the collection, analysis and exchange of information shaped and improved the project in many ways. From my perspective, in the area of Policy & Practices, two of the team’s accomplishments stand out:

  1. The biosafety considerations and the inclusion of the safety device. Preventing the contamination of the environment with engineered bacteria is of utmost importance. To address this, bacteria would be made strictly dependent on the conditions in the bandaid. As an alternative, the team proposed the use of a cream containing the purified inhibitor; with no live bacteria around, this would completely eliminate the possibility of contamination.
  2. The policy brief proposing a change in the distribution of funds between research and marketing in the biopharmaceutical industry. Marketing overspends to the detriment of research, the team argued, and the society would benefit greatly if this situation changed.

Here is a digram of the Policy & Practices component. Arrows show direction and extent of information flow.

Slide1

FINAL WORD

I’m thinking how this was a high-school team which couldn’t have had much synthetic biology knowledge and experience. Also, how they only had one year to do the project, and how this project was just one of many things they did at school. I’m so impressed! Aren’t you?

And I wonder…

What made this team exceptional?

What would it take to get this kind of rich, quality learning into more schools?

 

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