Introduction

As the population expands, nutrition has become a growing concern. Vitamins B9 and B12 specifically are both unable to be synthesized by humans and play a critical role in growth and development. Vitamin B9, or folate, is necessary for the production and maintenance of new cells, DNA and RNA synthesis, and for preventing changes in DNA. Deficiency of vitamin B9 may cause anemia, glossitis, diarrhea, depression, confusion, and in pregnant women it can cause fetal neural tube defects as well as other brain defects. Vitamin B12 is critical for normal function of the brain and nervous system due to its role in myelin synthesis. Deficiency of vitamin B12 may cause irreversible damage to the brain and nervous system. For these reasons, regulation of vitamin B9 and B12 concentrations is potentially useful for applications like manufacturing sensors for the detection of deficiency.

The growing importance of nutrition in genetic engineering may lead towards interesting synthetic biology applications of systems that deal with vitamins B9 and B12 in the realms of nutrition, manufacturing, and diagnostics. However, if synthetic systems are to be made to interact with these compounds -folate and cobalamin- means must be developed to regulate expression in response to the presence of these within the system. Riboswitches are one such method of regulation, but at the translational level. Riboswitches are strands of RNA that form conformations in response to the absence or presence of an aptamer. These conformation restrict the ability of the ribosome to bind to the mRNA, which prevents translation and thus gene expression. Naturally occurring riboswitches exist for both vitamins B9 and B12 which may be useful when attempting to regulate systems that interact with these aptamers.

Project Inspiration

A common concern among pregnant women all over the world is developing gestational deficiencies in vitamins B9 and B12. Lacking these essential nutrients during pregnancy can lead to birth defects such as spina bifida and other neural tube defects. Unfortunately, due to both lack of proper nutrient education and access to the current blood tests, pregnant women and their children in developing countries often unknowingly suffer from these specific deficiencies. Our project is aimed to increase the accessibility of vitamins B9 and B12 preliminary screening. The microbial method we are investigating would require less infrastructure, equipment, and time than the current method involving blood work.

To produce the screening test, the Rose-Hulman Institute of Technology iGEM team plans to engineer E. coli to express mRNA constitutively, regulated by riboswitches for vitamin B9, B12, and derivatives. Conformational changes of these riboswitches are induced by complexing with these substrate molecules which activates or deactivates the ability of the mRNA to be translated. When the substrate is bound to the riboswitch, expression of the chromoprotein reporter is repressed. This process will provide a visible indicator of a deficiency, which does not require sophisticated equipment to analyze. Additionally, the final system is intended to be produced for a portable cup or strip chassis, with which the genetic system would be easily transported and stored for use.