Vitamin B12 (VB12) deficiency is a common and prevalent condition which can engender neurological and hematological abnormalities. However, current measurements of VB12 level are usually erratic and lack sensitivity. Here, we present a portable and easy-to-use bioelectronic sensor for assessing the VB12 level in the blood. This home health monitoring device is similar to a glucometer, which measures the blood sugar level within a drop of blood obtained via a finger prick. The biosensor produces energy in the presence of VB12 through a co-culture of engineered Escherichia coli (E. coli) and Shewanella oneidensis (S. oneidensis). To be more specific, the genetically modified E. coli carries a vitamin B12 riboswitch which controls the biosynthesis of lactate. The latter is subsequently metabolized by Shewanella oneidensis to produce a reducing potential. Shewanella oneidensis has the prodigious ability to reduce metal ions, so when it is in contact with an anode it is possible to create electricity.
I. The characteristics of Vitamin B12(B12)
Vitamin B12 deficiency can be considered a global public health problem as it affects millions of individuals. Deficiency in vitamin B12 can cause a wide range of hematological, gastrointestinal, psychiatric, and neurological disorders. In the worst cases of vitamin B12 deficiency people can suffer from deep depression, paranoia, delusions, memory loss, incontinence, loss of taste and smell, and more (Harvard health publishing, 2020).
Vitamin B12, also known as cobalamin, is a nutrient totally dependent on dietary sources especially in animal products such as meat, fish, eggs, and dairy (Romain, 2016). Therefore, vegetarians and vegans typically lack vitamin B12, which make them particularly at risk for disorders mentioned above.
Structure of vitamin B12
Vitamin B12 is a macromolecule constituted of four pyrrole molecules organised in a corrine kernel. In the center of the kernel is a cobalt atom. This atom is itself linked to a benzimidazole-ribose-phosphoric acid structure. The cobalt atom placed in the center can also be linked to different substituents, such as a cyanide, a hydroxyl group, a methyl group or an adenosyl residue (5-deoxyadenosyl). Depending on which substituent is linked to the central atom, different forms of vitamins are built (Guilland, 2013).
Figure 1: Structure of vitamin B12
Vitamin B12 (or Cobalamin) exits in nine different forms, four of those being predominant in the organism: cyanocobalamin, hydroxocobalamin, methylcobalamin and adenosylcobalamin. Methylcobalamin and adenosylcobalamin are the active forms of vitamine B12 and are co-factors for the enzymes methionine synthase and methylmalonyl-CoA mutase, respectively. To be absorbed by the body, cobalamin binds to a transporter, at this stage it is then considered as Transcobalamin and can therefore enter in the cell to be used in different metabolism (Guilland, 2013).
The physiological functions of vitamin B12
Vitamin B12 is vital in several aspects for the human body, notably for developing a proper nervous system and its functioning. Vitamin B12 participates in the synthesis of DNA and in the haematopoiesis, as well as the integrity of the digestive epithelium. Moreover, vitamin B12 is necessary for the development and the myelination of the central nervous system which explain that lack of B12 results in neurological disorder. (Oh, 2003)
II. The biosensors: A Riboswitch-Based Inducible Gene Expression
Riboswitches (RS) are RNA-based sensors that were discovered in 2002 (Serganov, 2013). They can bind to a variety of ions and molecules and exert a regulatory control of transcription, translation, splicing and RNA stability. Figure 2 shows the different transcription regulatory controls exerted by riboswitches (Serganov, 2013).
The cobalamin riboswitch is a translation inhibition riboswitch that uses cobalamin, or vitamin B12, as a ligand. It is found in many different bacteria such as E. coli. Upon binding, the riboswitch changes in conformation to hide the Ribosome binding site (RBS) and therefore inhibit translation as shown in figure 3.
Why use in particular the cobalamin riboswitch?
The ligand-specificity of the cobalamin riboswitch makes it a great structure for the development of a biosensor to detect cobalamin. As it works as a repressor of translation, an inverted activation construct has to be made to activate our electronic reporter system. To do so, a Tet repressor protein must be placed after the riboswitch.
Tet repressor proteins are regulatory proteins that repress transcription upon binding to a Tet operator sequence, placed upstream a Gene of Interest (GOI). By using such a system, we can trigger the expression of our GOI upon binding of cobalamin on the riboswitch as shown in figure 4.
- No binding on RS (Off state) → translation of TetR → binding of TetR on Tet operator → repression of GOI
- Binding on RS (On state) → Repression of TetR translation → No binding of TetR on Tet operator → De-repression of GOI
III. Lactate dehydrogenase expression, our gene of interest
The lactate dehydrogenase is the enzyme responsible for the lactic fermentation pathway. In our project the lactate dehydrogenase is used to produce lactate. The exact utilization of the enzyme and its metabolite (lactate) will be explained further.
It exists in human but also in E. coli. It is a quaternary protein, composed of two subunits M and H combined into four subunits organization (see Figure 5).
This enzyme catalyses the conversion of lactate into pyruvate, with the co-factor NAD+/NADH, H+. It also catalyses the reverse reaction (see figure 6).
IV. Production of Electricity : Shewanella oneidensis
Shewanella oneidensis (S. oneidensis) is an anaerobic facultative bacterium known for its ability to reduce metals. Such bacteria can therefore produce a measurable electrical signal if the metal used is the anode of an amperemeter. For our project, we decided to focus on the MET producing pathway (shown in Figure 7) that uses metabolites to generate a reduction power (Mao, 2014). The metabolite we decide to use is the Lactate because it is one of the preferential metabolites used by S. oneidensis to reduce metals.
In 2019, a novel bioelectronic reporter system using S. oneidensis was designed and tested. Scientists proved that using a co-culture system with E. coli (production of lactate) and S. oneidensis (electricity production) could be a promising alternative to traditional reporter systems (Zeng, 2019). And this is where our project start !
V. Cobatect - in a few sentence
In order to determine vitamin B12 levels within the bloodstream, cobatect is developing an at home detection device that does not require any professional assistance.
The idea was to use the co-culture of E. coli and S. oneidensis to develop a bioelectronic reporter system sensing level of vitamin B12 in a sample. The aim of cobatect is to engineer E. coli by integrating a cobalamin riboswitch, that activates the expression of lactate dehydrogenase. Consequently, the production of lactate is promoted in the presence of vitamin B12.
At this point, S. oneidensis, intervene by using the produced lactate to reduce ions. By putting an anode in the co-culture, S. oneidensis will produce an electric signal proportional to the level of lactate. The lactate itself should be produced proportionally to the level of B12.
Consequently, the intensity of the electric signal should be proportional to the level of vitamin B12. The intensity then measured, will correspond to a certain level of B12. By comparing the normal level of B12 in the bloodstream a person should have regarding his physical condition (height, weight, gender, age) to the level measured we will be able to indicate if the patient suffer from a deficiency or not.
VI. The device
One of our goals for Cobatect was to develop an easy-to-use miniaturized device like those which measure the glycemia. We imagined a device shown in Figure 9 that consists of two parts:
- An easily removable “cassette” with the blood collection tube that is in contact with the bacterial co-culture. With the co-culture there
is the anode that will transfer the electric signal initiated by S. oneidensis to the ammeter to record it.
- A “box” which contains the wire connected to the anode, and an electronic chip (stm32) that will serve as an ammeter, then save the information,
analyse it and upload it on a mobile application or just on a screen on the device.
The “cassette” will be disposable
The “box” should be kept for several utilizations.
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