MSU's Proposal

Shewlock in Water Treatment:

The ability to measure electricity production by Shewanella has the potential to be used as an economical alternative to current measurement systems that detect for harmful water contaminants.

Why?

As a Michigan team, water quality is an issue that hits very close to home. The Flint Water Crisisgained national attention for the extremely elevated lead contamination in the drinking water. This crisis began in 2014, however the Flint residents are advised to continue using filtered or bottled water until the pipes are fully replaced by the year 2020. Another famous American case of poor water quality is the Hinkley groundwater contamination made public by Erin Brockovich in 1993.

More

Microbial fuel cells (MFC’s) and other bioelectrochemical systems (BES) have been used in cyclic voltammetry 3 experiments for biosensing purposes and many environmental applications such as wastewater treatment 4 . Glass bioreactors and three-electrode systems have been widely used 5-7 . For example, Yang et al. used a platinum wire counter electrode, a carbon cloth working electrode, and a saturated calomel electrode (SCE) as a reference 5 . Single- chambered bioelectrochemical cells have been used to study electric current production in the bacteria known as Pseudomonas aeruginosa and Shewanella oneidensis MR-1 which have been utilized to create biosensors 6-7 . Cost-effective and easy-assembly BES would allow for easier in- field and environmental testing of water contaminants to utilize these biosensors on a broader scale and be used in wastewater treatment.

A simpler and more economical design for single-chambered bioelectrochemical cells was created in order to test the electricity production of S. oneidensis MR-1 ΔmtrB. The Mtr pathway in S. oneidensis MR-1 is an external electron transport pathway that transfers electrons to an external acceptor such as an anode, generating electric current that is utilized in bioelectrochemical systems. The mtrB gene was removed to prevent electron flow to an outside source through this key protein. Different plasmids were then inserted into this strain with the mtrB gene under control of a promoter that activates transcription of the protein when induced by compounds such as pesticides and metals in the water. Some of the various strains were ΔmtrB, ΔmtrB prL814, and ΔmtrB prL814-mtrB. These three strains were induced by IPTG which is an analog to allolactose that induces the lac operon in the respective plasmids which were under control of the T7A1 promoter. Later, additional strains were created by cutting out the T7A1 promoter out of the ΔmtrB prL814-mtrB strain and inserting Cu, NO 3 , K, Zn, Mo, Biotin, Blue Light, and Paraquat promoters. These different strains were inoculated into the newly designed single-chambered BES made from Mason glass jars and induced to then test for electricity production using cyclic voltammetry techniques with a potentiostat.

The advantages of using this method to create a biosensor over others, such as an optical biosensor using fluorescence 8 or a nucleic acid biosensor 9 , are not having to utilize expensive enzymes as in the optical biosensor or expensive equipment and the low demand of nucleic acids biosensors. These single-chambered BES made out of common household glass jars are a more cost-effective way to create a biosensor that can be utilized in a laboratory setting, industry, or in the field.

Mtr Pathway

The mtrB pathway harnesses electrons through metabolized lactate and transports the energy from mtrA to mtrC which is then capable of donating the electron to an external acceptor molecule. MtrB is the bridge between mtrA and mtrC, allowing the contact necessary for electron transport. In the absence of mtrB, it is predicted that mtrA will no longer associate with mtrC and therefore will hinder the external electron transport pathway.

Mtr External Electron Transportation pathway in S. oneidensis MR-1 Lactate dehydrogenase metabolizes lactate to pyruvate, transferring the released electrons to a quinone cycle in the membrane. CymA, coordinated the transfer of electrons from the quinone pool to mtrA. MtrB facilitates the interaction between mtrA and mtrC as the electrons flow to mtrC where they are accepted by an external molecule.

A New Measurement System

After running our project for quite some time we had started using current production instead of GFP fluorescence for protein expression. We realized that current measurements were many times faster with reproducible increase in current 18 minutes after induction with IPTG. This brought us to the realization that we could use current instead of GFP as a reporter for protein expression. We also realized that anyone could use this, that it would be faster than GFP, and it would be just as accurate. This method has many benefits over GFP fluorescence. The expression can be taken in anaerobic conditions, where the superfolder proteins in GFP need oxygen to fold properly. Current is a much faster method with induction visible after 18 minutes as opposed to multiple hours for GFP. Current requires less expensive equipment than GFP and current measurement has a continuous display while GFP only has intermittent results. All of these claims are visible in either the results tab of or the model tab. Due to all of these benefits to using current as a measurement tool we posit that current is a more effective measurement tool of protein expression than GFP fluorescence.