We truly believe that our project can make a great impact on the world. Throughout our summer preparation, we realized that to make our project more complete and all-rounded, we will need more time for the lab work. We have listed the possible future plans for our project.
We split our plans into two major parts - p-Cresol accumulation reduction and blood biomarker detection.
p-Cresol Accumulation Reduction
After several attempts of constructing the yebF-CBM-B plasmid all ended up in frameshift mutation, we propose that this recombinant protein is toxic to E. coli itself. However, T7 polymerase driving CBM-B can be tolerated in E. coli BL21(DE3), showing that E. coli is able to heterogeneously express large amounts of this bacteriocin protein.
Although purified bacteriocin can show significant inhibition of Clostridium growth, purified protein is not consistent with the goal of carrying forward the idea of live therapeutic. Hence, we will continue to try out other secretion systems inside E. coli in the future such as hylA secretion tag or Sec-/SRP- pathway to secrete the bacteriocin out of the cell.
Tyrosine ammonia-lyase & tyrosine transporter
According to FDA Guidance on Live Biotherapeutic Organisms (docket number FDA-2010-D-0500), live therapeutics should not include antibiotic resistance genes as selection markers. All genetic manipulation must be fixed on the bacterial chromosome to prevent horizontal genetic transfer. We were inspired by research published in Nature Biotech, that also uses E. coli Nissle as a chassis to manipulate metabolic pathways inside the human gut. They knocked-in the target genes using lambda Red recombineering into five different locations in E. coli Nissle’s chromosome. The finalized Cresolve bacteria will have the optimized TAL and tyrP gene inserted into the chromosome.
Also, there is still potential for the tyrosine degradation pathway. Research paper and iGEM Uppsala 2013 have reported that tyrosine ammonia-lyase should have a higher expression level in E. coli and thus able to consume higher amounts of tyrosine. We will further optimize the combination of promoters and ribosome binding sites for the enzyme involved in the tyrosine degradation pathway to increase the effectiveness of CreSolve.
Additional concerns towards E. coli Nissle
When researching uremic toxin, we found that most E. coli strains carry tnaA gene, which encodes for tryptophanase, that is able to convert tryptophan into indole, one of the uremic toxins. So, if we are using E. coli Nissle to solve uremic toxin accumulation in CKD patients, we have to acknowledge this problem. The best way is to knock out the tnaA gene. However, during the summer, we are unable to construct a double mutant strain E. coli Nissle tnaA- can- due to limited time. We will continue this work in the future.
Blood Biomarker Detection
p-Cresol sensing bacteria
Although this year we did not successfully construct a bacteria that is able to sense p-Cresol using the pchR regulon from Pseudomonas fluorescens PC24, there are other constructs, such as dmpR regulon, that have been proposed by iGEM Evry 2014 that are able to sense phenolic compounds. Research also has proven that dmpR can sense p-Cresol. We may incorporate these constructs into our CreSense bio-sensing platform, but the specificity of these sensing regulons is still of concern to us.
Oh My Gut project will be developed into a start-up company, named CreSolution Biotechnologies. CreSolution Biotechnologies will focus on 4 divisions, which are Living Therapeutics Division, Medical Diagnosis Device Division, CreSolution Cloud Services and will develop into CreSolution Clinic. CreSolution Biotechnologies aims to expand the area served to cover most of the countries around the world, starting by entering the United States, China and India, which represent the largest markets in the world. Further expansion will focus on the European Union and other Asia Pacific countries. CreSolution Biotechnologies aspire to change the public’s perspective on GMO, synthetic biology and genetic engineering, while providing solutions to improve and extend the lives of people by setting up an affordable healthcare platform to increase the accessibility to ANYONE who requires medical advice, attention and products.
- Kleiner-Grote, G. R. M., Risse, J. M., & Friehs, K. (2018). Secretion of recombinant proteins from E. coli. Engineering in Life Sciences, 18(8), 532–550.
- Early Clinical Trials with Live Biotherapeutic Products ... (n.d.). Retrieved from https://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/General/UCM292704.pdf.
- Isabella, V. M., Ha, B. N., Castillo, M. J., Lubkowicz, D. J., Rowe, S. E., Millet, Y. A., … Falb, D. (2018). Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nature Biotechnology, 36(9), 857–864.
- Jendresen, C. B., Stahlhut, S. G., Li, M., Gaspar, P., Siedler, S., Förster, J., … Nielsen, A. T. (2015). Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae. Applied and Environmental Microbiology, 81(13), 4458–4476.
- Sarand, I., Skarfstad, E., Forsman, M., Romantschuk, M., & Shingler, V. (2001). Role of the dmpR-Mediated Regulatory Circuit in Bacterial Biodegradation Properties in Methylphenol-Amended Soils. Applied and Environmental Microbiology, 67(1), 162–171.