Our project is a dCas9 powered gene circuit that induces a gene only in the presence of a known DNA sequence. However, we have chosen to showcase this construct in the context of a pressing problem in medicine: antibiotic resistance. We envision a system where the introduction neutral stimulus could produce a negative pressure on bacteria with antibiotic resistance via activation of a programmable cell death gene. The process could move existing bacterial populations away from antibiotic resistance when not using antibiotics.

We hope to perform a proof of concept experiment to test whether the presence of a plasmid can activate a reporter gene. We intend to mark initial trials with fluorescent proteins in order to easily isolate the effect of the circuit on transcription but then to extend the effect to the study of population growth patterns.

How did we improve biohazard safety?

  • We used DH5-α strain of E. Coli, which has poor pathogenicity owing to its lack of genes coding for invasion, adhesion, enterotoxins, as well as long-chain lipopolysacharides
  • Studies show that this strain is unlikely to survive in mouse guts, and therefore, pathogenicity towards humans is unlikely [1]
  • Worked exclusively with Risk Group 1 organisms, as per iGEM regulations, in a Biosafety Level 2 laboratory with well-defined protocols for disposal of chemical and biological waste
  • Each team member took the Duke University safety courses in lab safety and biohazard safety; a link to policy overviews can be found here.
  • Included general lab procedures such as use of PPE, sharps, fume hoods, proper chemical and biological waste disposal, as well as emergency procedures
  • Whenever potentially noxious chemicals are handled, they are done so in a chemical fume hood erring on the side of caution and gloves are changed immediately after handling potentially harmful chemicals such as Ethidium Bromide

Risks in our project?

  • The use of bacteria which possess antibiotic resistance (even if only to ampicillin or chloramphenicol) pose a potential environmental risk
  • If genes coding for resistance were passed to or picked up by pathogenic species of bacteria, the potential for danger arises, which is why we took precautions to ensure that bacteria were safely disposed of
  • Our project, if studied to completion, might provide a way to parse through a population and remove undesirable individuals (i.e. bacteria which possess antibiotic resistance), potentially bypassing the growing antibiotic resistance problems
  • However, the potential exists for someone to modify the gene circuit to remove healthy cells or to kill human cells
  • Given that the method for “removing” individuals is lysis, this would result in exogenous genetic material, which if left without nuclease activity, could be transformed into another cell
  • If benign bacteria with antibiotic resistance were to pick up this material, they might be adversely affected by the gene, which could detriment microbiomes
  • The project, in its current state, is still in a proof-of-concept stage, and as such, poses few safety threats, but if it were to be modified for use in other more pathogenic bacteria, cancer cells, or viruses, we would need to consider more precautions for future development

Any long term applications in microbial populations within humans should place safety as the primary concern. As such, the cell death gene would need to be with minimized side effects on the patient. Further, the long term successful implementation would work on a largely population level, so the spread of the antibiotic detecting plasmid would be important to the overall phenotypic change away from antibiotic resistance. But particular care would be necessary to limit unwanted infection of synthetic plasmids into those who do not want the plasmid. Care should also be taken to avoid any pathogenicity that could arise from the added plasmid and cell death gene.

Cysteine-Deleted Protegrin I

  • Initially, we tested Protegrin I for antibacterial properties, and for use as a potential “kill switch” to be implemented in the main project
  • However, Protegrin I has hemolytic properties, and as such, would have been unfit for use on humans or on model organisms like mice
  • Literature suggests that removing cysteine residues can disrupt disulfide bridge formation in the protein, reducing hemolytic capacity, but still maintaining broad spectrum antibacterial activity [2]
  • In order to improve the safety of using Protegrin I in future antibacterial studies, we developed a biobrick which essentially is Protegrin I minus four cysteine residues
  • However, the hemolytic capacity of the biobricked form of Protegrin I is untested, and will require verification in order to determine its “safety” towards humans