How have we contributed to the work of future iGEM teams?

Provided critical examination of recent research on IBD and therapeutics, as well as past iGEM projects

For a therapeutic project on IBD, it is necessary to examine current research, treatment, as well as proposals. We not only summarized our findings from literature in our Wiki but also summarized all past iGEM teams’ work that involved IBD diagnosis or therapeutics. These can provide guidance for future teams to note trends as well as evaluate novelty of their new ideas. Alternatively, our past iGEM team table can also provide future teams with directions for collaboration opportunities and areas of improvement.

See Background and Rationale for more information.

Created both basic and composite parts for the targeted elimination of AIEC bacteria

The positive feedback system for signal amplification

Many past iGEM teams who focused on IBD therapeutics or diagnosis chose nitric oxide (NO) as a biomarker of gut inflammation. However, the limited half-life of NO due to oxidation has rarely been considered in their design process. This year, we designed composite parts BBa_K4010007 and BBa_K4010010 that can activate genes downstream of a flhDC promoter not only upon sensing both autoinducer-3 and NO but also maintain that signal even after NO has been oxidized to nitrate. Specifically, we took advantage of dual regulation by both NsrR and NarL on the PyeaR promoter and integrated a positive feedback design to amplify the response from the initial encounter with NO.

While many parts of this design are tailored to our circuits, we made many contributions to the iGEM Parts Registry for future teams to use or improve upon. The basic part BBa_K4010006 coding sequence for the NarX histidine kinase sensor has been added to ensure future iGEM teams can create composite parts that involve the NarX/L two component regulatory system. As well, we not only included the overall design composite part but also divided the histidine kinase and response regulator modules into individual composite parts BBa_K4010008, BBa_K4010009, BBa_K4010011, and BBa_K4010012. This would allow future iGEM teams to pick and choose components within our design to suit their purposes, such as using a secondary biomarker other than autoinducer-3 by switching out the QseBC and flhDC combination for another two-component regulatory system.

TorA transportation signals for the release of colicin without inducing bacteria cell lysis

Previous projects using colicin released the bacteriocin via cell lysis. However, in the composite parts BBa_K4010004 and BBa_K4010005, a TorA transportation signal is attached at the N-terminal. As such, colicins E1 and E9 can be transported across the host bacterial inner membrane without killing the host cell, which increases the exportation of colicin over a longer period of time. This method of circumventing cell lysis can increase the efficiency of future iGEM projects creating bacteriocin-based probiotics.

To create these composite parts, parts BBa_K822002 was separated into colicin E1 and the immunity protein, which allows for increased modularity. This allows more design flexibility for future iGEM teams in incorporating the bacteriocins. The parts for colicin E1 (BBa_K822002) and colicin E9 (BBa_K242201) was also modified to be His-tagged in the new parts BBa_K4010001 and BBa_K4010003 to facilitate the protein purification process for future teams.

Basic Parts

See Parts for more information.

Critically analyzed various probiotic bacterial strains and provided recommendations for future iGEM teams

Under the guidance of our advisor, we saw the importance of selecting a chassis to implement our project into a patient’s treatment plan. Specifically, we chose four candidate bacteria families or strains based on their safety profile or therapeutic potentials: E.coli Nissle 1917, Lactobacillus, Bifidobacterium, and Streptococcus thermophilus. We highlighted main considerations such as colonization ability, immunogenicity, ease of culturing, and overall safety profile. Surprisingly, under the guidance of an established professor and researcher at McMaster, we found E.coli Nissle 1917 in past research studies to have a concerning genotoxicity potential through colibactin production despite its well characterization and popularity among past iGEM teams. While this is still under heavy debate, we determined that future teams should be advised to exercise caution upon selecting their chassis, especially if their projects are therapeutic in nature. Our table will not only provide them with our findings in literature but also provide a template for their chassis evaluation.

See Implementation for more information.

Presented experiments for verifying the killing of AIEC bacteria

In addition to co-culture assays, detailed protocols for XTT and crystal violet assays for quantifying the number of E.coli were included to measure bacteria in biofilms both in the living and dead state. Furthermore, these experiments estimate the relative number of a specific bacteria even in a culture of two different species by mathematically eliminating the background from bacteria the researchers are not interested in. This has not been described in detail by most literature, and would help future teams measuring the killing or growth of numerous biofilm-forming bacteria.

See Engineering for more information.

McMaster SynBio 2021