Team:Alma/Design
Designing our Solution: Plaque Attack
The prior research done by our team (specifically by Brianna Ricker) helped to define the scope and importance of the problem. We also sought expert feedback to help better understand what would be needed to design a solution. Below, you will find more information about our design process, including background on the metabolic pathway our solution relies on, and the design of our initial BioBricks, and our designs for a safety switch.
TMA Metabolism in Methanogens
In humans, the life-cycle of TMAO begins with the synthesis of trimethylamine (TMA) by bacteria in the gut. TMA enters the bloodstream where it is then transported to the liver and oxidized to form TMAO. TMAO reenters the bloodstream where it can wreak havoc on vascular walls. This cycle presents several possible treatment avenues, one of which is an alteration of the gut microbiota to reduce the amount of TMA entering the bloodstream. One possible route proposed by Brugere et al. is to metabolize the TMA produced in the gut into methane using another strain of bacteria. Brugere et al. continued to describe a bacterial group called the methanogens, which contains several strains of bacteria harboring genes that enable it to convert methylamines and TMA into methane gas. Although these methanogens are present in healthy gut microbiota, they are outcompeted by more aggressive bacterial strains, and are too few in number to metabolize TMA.
However, synthetic biology and genetic engineering could allow the genes of interest to be placed into a more appropriate chassis. The gut microbiome in a healthy individual normally consists of five phyla; Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Cerrucomicrobia. The specific composition of species varies between individuals, but Bacteroidetes and Firmicutes make up >90% of the species within the gut microbiome in a healthy small intestine. E. coli represents a good starting strain; it is not a highly aggressive colonizer of the gut, but it is still present frequently enough to produce an effect, and it is well studied and has shown great potential for genetic modification in countless other studies.
The genes that allow methanogens to breakdown TMA into methane gas include MttB—trimethylamine transferase, MttC—trimethylamine corrinoid protein, and MtbA—methylcobamide: CoM methyltransferase. The synthesis of trimethylamine transferase requires the addition of the non-standard amino acid pyrrolysine; to accomplish this, proteins capable of synthesizing pyrrolysine must also be incorporated into the engineered bacterium. These genes include PylB—3-methylornithine synthase, PylC—3-methylornithine-L-lysine ligase, PylD—3-methlyornithyl-N6-L-lysine dehydrogenase, and PylS—pyrrolysine-tRNA ligase. Finally, the appropriate transfer RNA, encoded by PylT, must also be present.
As shown in the above diagram from Brugere et al (to the left), once MttB has the pyrrolysine incorporated, it will remove a methyl group from TMA to generate dimethylamine, or DMA. This enzyme does not release this group as methane, but rather transferase it to the associated corrinoid protein, MttC. The exact mechanism for this step is unknown – whether or not the corrinoid protein must be first bound is unclear. MtbA can then remove the methyl from MttC’s corrnoid group and transfer it to coenzyme M, whereby it can enter into the activated methyl cycle of an organism’s metabolism.
Designing the Initial BioBricks
To begin to construct our TMA-degrading device, we first attempted to clone each gene as an expression cassette. To do this, we needed the protein sequence of each gene (or the RNA sequence, in the case of PylT). Fortunately, these were readily available, and the sequences for all genes were present in at least one organism - Methanogenic archaeon ISO4-H5. The amino acid sequences for these genes that we obtained are available for download as a PDF. Note that this contains both the primary amino acid sequences and the original nucleotide sequence from that organism.
We then reverse translated the amino acid sequences in a way that ensured: 1) compliance with BioBrick standards, 2) easy synthesis by IDT – no repetitive sequences and 3) codon optimization for E. coli, as long as this did not interfere with the first two criteria. This was accomplished using a Java program written by the 2016 Kingsborough iGEM team – a simple tool called protein2bioBrick. The resulting sequences we created are also available to download.
Our further design combines several of these BioBricks together to build an Operon, such as one containing MttB and MttC expressed in tandem (at the same levels - see the next section for a discussion of relative amounts of these two proteins). These Operons would be built in successive steps by cloning each protein generating unit (RBS and Gene) under the control of a common promoter, such as BBa_R0011 or BBa_R0040, which would allow inducible or constitutive expression depending on the genetic background of the cell.
Designing a Cell-Free Solution
Our main design involved placing at least 7 genes inside one organisms - 3 for the metabolic activity, and 4 to support the incorporation of pyrrolysine into MttB. Such an approach could be unwieldy and present unexpected problems. In addition, we learned from our expert feedback that there would be both positives - long lasting treatment - and negatives - safety concerns with GMOs inside the body - associated with our design.
We therefore decided to explore different cell-free systems, based upon what is known about the action of the 3 metabolic enzymes we were working with: MttB, MttC, and MtbA.
Although the enzymes involved in TMA metabolism are not well characterized, the current understanding is that MttB binds to MttC and TMA in some order to transfer the methyl group from TMA to MttC. Then, MttC is recycled by the action of MtbA, which moves the methyl group to coenzyme M. We attempted to model this behavior to better understand how we should construct an Operon and the cell-free system - this is shown on the left, in which B is MttB, C is MttC, TMA is the substrate, D is DMA (one of the products), and bA is MtbA. The star represents the transfered methyl group. Kf is a reaction rate constant which we wanted to either find, derive, or estimate.
It ended up being difficult to mathematically model this system without experimentally derived enzyme kinetic parameters. We even consulted with Dr. James Mazzuca to determine if this could be predicted from the crystal structure of MttB alone (PDB ID = 4YYC). However, it turns out that even that crystal structure provided limited information, since it was derived by accident!
Our cell-free system would work by having MttB and MttC appended with a varying number of the SpyCatcher and SpyTag sequences. These proteins can form isopeptide bonds when mixed, and we hoped that this would allow the MttB-MttC fusion to travel in the gut as a single molecule, eliminating the need to answer questions regarding the binding affinity of these two proteins for each other and how they could diffuse separately in the gut. These fusion proteins would be separately produced in a cell-free transcription-translation system (myTXTL from Arbor Biosciences), mixed to form the isopeptide bond, and then delivered to a patient in a pill. In our cell-free system, there would be no recycling of MttC, so the amount of fusion protein that is delivered will capture TMA at a 1:1 ratio (ideally).
Designing a Safety Switch
To make our solution effective, it also needs to be safe. The bacteria we introduce should be able to survive the trip through the stomach into the intestines, and should be present only in the areas of the small intestines where the TMA-producing bacteria are present (and where TMA is absorbed). We also want to ensure that our modified bacteria does not cause harm by either travelling to other parts of the body or in the environment (if it exits the gut through the colon).
Thus, we considered creating a number of 'safety-switches' or 'kill-switches' that could control the bacteria's behavior and initiate a self-destruct if it wandered from the intended target. These included designs for a light sensitive kill switch: since the inside of the intestines is dark, this would be a measure to prevent growth of any bacteria that escapes the body. This kill switch was designed to work using BioBrick K592016. Similar kill-switches could be created that respond to temperature (inside the body is warmer than most outside environments) and phosphate levels. These designs can even be used in combination for a higher degree of safety and control. However, we decided to start with a pH sensitive kill switch, since it also could allow us to localize the bacteria to a particular part of the intestines.
We would want to target our bacteria to the proximal region of the small intestines - so we would need bacteria that can thrive in a slightly acidic environment. These bacteria would be encapsulated to protect them in their journey through the stomach, released only when reaching the small intestines. To prevent growth of the bacteria that escape prematurely, we would want to build a low-pH sensitive kill switch. A promoter system for creating such a device already exists: BBa_K1231000. With some fine tuning, this can also help destroy any bacteria that make it to the ascending colon.
To gain further control over the system, we decided to pair the above promoter and intended kill-switch with the inverse - a high pH promoter and kill-switch. We did not see a convenient part to play this role, so we sought to design one ourself, by placing an inverter after the low-pH promoter. If combined with the previous part in a specialized circuit, we may be able to localize the bacteria to a particular part of the intestines. We sought to test this design for a high-pH promoter by first seeing how it would drive expression of a reporter gene such as RFP - you can learn more about our design for this BioBrick, BBa_K2905016, elsewhere on our site or on the registry.