Team:Virgina/Safety
Safety
This goal of this project is to design a bacterial chassis that, when inserted into the human digestive system, possess the capacity to moderate post-prandial glycemic spikes via the absorption and polymerization of free floating simple sugars. In order for this to be a viable product for human consumption, a variety of safety measures must be considered and implemented to ensure that the product is incapable of harming the host. These factors include, but are not limited to: the biosafety level of the both the chassis and its transfected genes, the potential impact on blood glucose levels, and the removal and elimination of the product.
Biosafety
Due to the invasive nature of this project, the safety level of the organism being inserted, as well as its transfected genes, must be analyzed. The chassis used for this experiment is a strain of E. coli known as K12. K12 E. coli is a Biosafety Level 1 organism, therefore it is not known to cause disease in humans and presents a low risk to individuals working with it. In order for the bacterial chassis to moderate levels of blood glucose, the gene EIIA (BBa_K1508003) was used in one of the two biobricks assembled for this project. EIIA is a Biosafety Level 1 gene that codes for the increased uptake of glucose. Two additional genes inserted into the K12 chassis are glgC (BBa_K118018) and sacB (BBa_K322921). glgC functions to polymerize glucose into glycogen, while sacB functions to polymerize fructose into levan. Overall, the chassis and the genes transfected into it separately confer a biosafety level of 1 for this project.
Blood Glucose Levels
As previously stated, the goal of this project is to mitigate glycemic spikes through the absorption of excess glucose. Assays performed on glgC indicate that K12 cells expressing this gene polymerize high levels of glucose. This means that the presence of these cells in the intestinal lumen will theoretically lower the amounts of free sugars that are able to be absorbed by the intestine. Currently, more experimentation is needed before we are able to analyze the effect of our chassis in vivo. However, because we have demonstrated that our modified K12 bacteria are able to carry out the conversion of glucose to glycogen, it is important to note that a potential side effect to introducing this into the human digestive system is hypoglycemia.
Removal and Elimination of the product
In order for this product to function as a treatment for hyperglycemic spikes, the amount of glucose absorbed as well as the duration of its functionality must be tightly regulated. Currently, our bacterial kill switch is the sacB gene. When exposed the fructose, this gene codes for levan, which continues to build up until it reaches a toxic level, killing the cell. Fructose assays performed on this gene indicate an inhibition of cell growth or lysis after two hours. This means that in order to eliminate this product from the digestive system, it must be consumed along with adequate levels of fructose to lyse the cells. A potential issue with this killswitch is that, as of right now, we are unable to determine how it will behave in vivo. For example, if the rate of fructose absorption through the intestine is much higher than the rate of absorption in the bacterium, then it could take anywhere between a few hours to a few days before the levels of intracellular levan is present at toxic levels in the bacteria. Additionally, if a mutation occurred that removed the functionality of sacB, there are no backup systems in place that would remove the bacterium from the system. This would lead to a replicating colony of bacterium actively competing for glucose in the gut. Depending on the functionality of these colonies in vivo, the results could lead hypoglycemia and potential interactions with the microbial flora already present in the digestive tract.