iGEM Lund 2019

Project Design

This page discusses the design of the project and the theory behind it.

Design Process

Our design followed the engineering principle of first analyzing the problem which had been chosen, that is, the accumulation of toxic metals in the body, and then analyzing a probable solution. The solution we came up with was to prevent accumulation of the toxic metals before they could act out their harmful effects in the body. Since a majority of in vivo accumulated metals comes from food and beverage, the idea of a modified probiotic bacteria emerged, which could, in theory, accumulate the metal in your place and then leave the body via excretion. By investigating literature we found bacteria which was capable of accumulating metals, which then could be used as a model bacteria and applied to a safe, ingestible bacteria. Genes for metal remediation were identified (specific details of the design and composition can be found below). Since most probiotic bacteria can be a struggle to work with, a probiotic strain of E. coli was chosen: E. coli Nissle 1917 (henceforth EcN). This strain isn't documented well, therefore E. coli BL21-DE3 was also used as a safe bet.

How do some Bacteria Deal with Toxic Metals?

Depending on the level of exposure of toxic metals bacteria have had during its evolution its genome has adapted in different ways. A special case is Cupriavidus metallidurans strain CH34, as it has been subject to high levels of toxic metal stress and has developed two megaplasmids, pMOL28 and pMOL30 because of it. These plasmids contain genes coding for toxic metal remediation.

Applied Design

By taking some of the genes from C. metallidurans CH34 and inserting them into a probiotic chassis, EcN, we could increase the arsenic- and lead-accumulation capabilities of EcN. The bacteria is then to be ingested as a probiotic, where it adapts to our gut microbiome due to the innate abilities of EcN. There, it will accumulate arsenic and lead in your place. Soon the bacteria full of metals will be excreted, decreasing the amount of toxic metals in your body. Our engineered EcN will most likely not cause any harm in nature. In fact, it will most likely die due to its wasteful expendidature of energy on overexpressing our proteins. However, we understand that while genetically modified microbes are rare, the idea of environmental exposure is frightening.

Lead

The proteins responsible for lead remediation in C. metallidurans are present in the pbr operon. The mechanism of the C. metallidurans pbr operon on pMOL30 is described in figure 1.

Figure 1: from “Lead(II) resistance in Cupriavidus metallidurans CH34: the interplay between plasmid and chromosomally-located functions” Safiyh Taghavi, Celine Lesaulnier, Sebastien Monchy, Ruddy Wattiez, Max Mergeay, Daniel van der Lelie

Since we’re focusing on bioaccumulation our interests are PbrT, a lead transporter protein, and PbrD, a putative lead-binding protein. Since we're interested in the highest amount of expression possible we’ve used a T7 promoter (BBa_I719005) for use in E. coli BL21-DE3, and Tac promoter (BBa_K3282006) for use in EcN.

pbrT: BBa_K3282001

pbrD: BBa_K3282002

Composite part with pbrT, pbrD and a T7 promoter: BBa_K3282005

Composite part with pbrT, pbrD and a Tac promoter: BBa_K3282007

Figure 2: An overview of the lead accumulation mechanism.

Arsenic

The ars operon is widespread amongst bacteria, including EcN. However, the accumulation capabilities of EcN is low, something we aimed to improve. A paper from 2004 by Kostal et al. showcased that overexpression of ArsR, a regulatory protein in the ars operon, in genetically modified E. coli, increased its accumulation capabilities by 5-60 fold. Applying the same logic we decided to include this protein as an accumulation protein. Because arsenic transport is facilitated in glycerol uptake facilitator proteins (GlpFs) we do not have to include a transport protein. Similarly, since we’re aiming for the highest possible expression and have thus chosen a T7 promoter (BBa_I719005) for use in E.coli BL21-DE3 and Tac-promoter (BBa_K3282006) for use in EcN.

arsR: BBa_K3282000

Composite part with arsR and a T7 promoter: BBa_K3282003

Composite part with arsR and a Tac promoter: BBa_K3282004

Figure 3: An overview of the arsenic accumulation mechanism.

Experimental Plan

The experiments were designed in a way to prove the protein expression and protein function. Before we could start experiments the proper constructs were created according to common biochemical methods of cloning and transformed into the correct strains. The experimental plan was made to first characterize the growth of the bacteria in their preferred conditions, and to test the expression of the proteins that had been implemented. This was done by growing cultures, testing cell growth and using SDS-PAGE to illustrate the presence of the expected protein. Thereafter, we needed to prove the concept and the function of the proteins. This was done by growing culture in toxic metal-infused media. The uptake of metal into the cell was investigated by measuring the concentration of metal in the media over time. We could then prove that accumulation of metals occured by a decreased concentration of metals in the media.

The plan was to then prove the function of the constructs in realistic conditions. This would be proved by letting earthworms consume the engineered EcN as well as toxic metals. This would be compared with a control group of worms that were supplied with toxic metals without the helpful bacteria. The proof of the concept in a realistic environment would then come from the survival rate of worms. Another option for the design of this experiment would be to measure the concentration of toxic metals in the actual worms. Worms fed with both metal and engineered EcN would according to our hypothesis have a lower concentration of metal than a control group of worms fed with only metal. Even though this experiment had several suggestions for design, there was not enough time to prove the concept in a realistic setting using worms. Instead, the conditions of the gut was simulated by lowering the pH of the media.

To see how it all played out, head over to the Results-page!