Team:ATOMS-Turkiye/HumanPractice:Safety

  Dear ATOMS-TÜRKİYE iGEM Team 2013,

I am Stewart Humble, a member of the iGEM Grenoble-EMSE-LSU Team for 2013.  I was asked to give a quick informal overview and comparison with our project and my previous research at LSU to address some of the biosafety issues and questions that may arise for our team.

As an undergraduate at LSU, I performed my thesis work on the synthesis and cellular investigations of a specific photosensitizer that is a chlorophyll derivative called Chlorin e6.  My work concentrated on the applications of this photosensitizer in the clinical process known as photodynamic therapy (PDT).  PDT is used to destroy tumors, and involves three key components:  a photosensitizer, light, and tissue oxygen.

By modifying this derivative through the addition of positively charged amino acids (lysine), I was able to give strengthen its amphiphilic characteristics.  Amphiphilicity is a measure of the extent to which a molecule can behave in both a polar and nonpolar way.  This gives these molecules the ability to dissolve in the blood and also to pass through hydrophobic cell membranes.  After biological studies, I found that the molecular photosensitizer I constructed was very potent in terms of phototoxicity, which means that even at very low concentration there was destruction of the cancer cells.

In terms of biosafety, I want to focus on the basic principles of biochemistry and cell-photosensitizer interaction.  In general, my photosensitizer was much more of a safety risk due to its ability to uptaken into individual cells from the extracellular space.  Additionally, it is not heavily altered by spending time in the extracellular space due to its amphiphilic characteristics. KillerRed, however, is a "genetic" photosensitizer that is created within a cell of interest and (as far as we know) can only act upon that individual cell.  This is due to the very unique fact that KillerRed is a protein that folds to form a chromophore that produces 10-fold more reactive oxygen species (ROS) than other fluorescent proteins such as GFP.  All proteins have unique foldings that can be quite sensitive to its surroundings (pH, temperature, etc.) and if a cell containing KillerRed were to lyse and release the photosensitizer, I believe that no other cells would be greatly (if al all) affected due to two main points:

1.)  The KillerRed protein may become denatured (unfolded) due to a change in environment, breaking apart the chromophore and rendering all ROS production to null

2.)  The KillerRed protein maintained its proper folding, but fails to enter any nearby cells due to its size (macromolecule) and inability to be uptaken into other cells (lack of receptors, etc to trigger any other form of uptake)     

I hope this explanation will help to address some of the safety concerns we will be collaborating upon for the iGEM Competition.  Please let our team know if anything further is needed.  Thanks!

Hi ATOMS Turkiye,

For the first question, we will introduce our gene construct to the bacteria before hand and then introduce it to the host bee via food (a process called paratransgenesis), so it is highly unlikely for the other bacteria to get this part. Also, our system is optimized for b. subtilis, the promoters we used and the products as a whole have slim to no chance of functioning anywhere else.

For the second question, the bacillus strain we selected is native to the bees' gut. It is normally not found in the honey stomach of the bee, even if the bacteria were to get into honey, it will still be in the natural levels. Other than that, according to literature, IPTG is never traced in honey, so our little germs, even if they can make it into honey, will be killed because of our kill switch.

The third question was a hard one because there is actually no real data about the p-coumaric acid levels let alone it is not known if such a thing as p-coumaric acid overdose exists. We used in our system a constitutive promoter for the production of it too. What we expect and what we could argue from literature is that having p-coumaric acid is in all cases preferable. Maybe exceeding levels could cause increased energy consumption and maybe even acid accumulation, a decrease in tyrosine(a precursor of p-coumaric acid) etc. What most probably will happen (and what we actually hope for) is that the natural feedback mechanism will be triggered and control the process. But you are right, a inductive system would have been prettier :)

For the last question, p-coumaric acid is mostly produced by plants. Even vanilla production and cinnamon flavour begins with p-coumaric acid. Many plants produce p-coumaric acid so p-coumaric acid exposure should not cause any harm to them. Besides, p-coumaric acid will be produced in the midgut of the bee, so it is unlikely for p-coumaric acid to find its way directly to the plant.

Kind regards,

Oguz Bolgi on behalf of METU-Turkey