Team:Yale/methods
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Developing a Framework for the Genetic Manipulation of Non-Model and Environmentally Significant Microbes
Project Strategy- Overview
Click on a component to learn more about our method.
Technique-Independent Procedures
Mage Implementation (Cyanobacteria)
CRISPR-Cas9 Implementation Pathway (Sinorhizobium)
Growth
Cyanobacterium
Biofilm formation on surfaces is an issue in the medical field, naval industry, and other areas. We developed an anti-fouling peptide with two modular components: a mussel adhesion protein (MAP) anchor and LL-37, an antimicrobial peptide. MAPs can selectively attach to metal and organic surfaces via L-3,5-dihydroxyphenylalanine (L-DOPA), a nonstandard amino acid that was incorporated using a genetically recoded organism (GRO). Because this peptide is toxic to the GRO in which it is produced, we designed a better controlled inducible system that limits basal expression. This was achieved through a novel T7 riboregulation system that controls expression at both the transcriptional and translational levels.
Transformation
Cyanobacterium
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Selection
Cyanobacterium
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Identifying Recombinases
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Since we intend to synthesize an anti-microbial peptide, it is possible that the peptide will be toxic to the E. coli used in our synthetic route. To improve our overall protein yield, we designed a plasmid with specific locks in place to control expression of the T7 RNA polymerase, an RNA polymerase from the T7 bacteriophage. When the T7 RNA polymerase is expressed, it can then specifically target the T7 promoter located in a different plasmid upstream of our coding sequence, initiating protein translation. The specific mechanism of our T7 riboregulation system is outlined in a section below.6,7 Back to Top
Gene Knockouts with Mage
FLP-FRT Recombination
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Since we intend to synthesize an anti-microbial peptide, it is possible that the peptide will be toxic to the E. coli used in our synthetic route. To improve our overall protein yield, we designed a plasmid with specific locks in place to control expression of the T7 RNA polymerase, an RNA polymerase from the T7 bacteriophage. When the T7 RNA polymerase is expressed, it can then specifically target the T7 promoter located in a different plasmid upstream of our coding sequence, initiating protein translation. The specific mechanism of our T7 riboregulation system is outlined in a section below.6,7 Back to Top
DNA Assemblies
If opportunity doesn't knock, build a door.
Gibson Assembly
Biofilm formation on surfaces is an issue in the medical field, naval industry, and other areas. We developed an anti-fouling peptide with two modular components: a mussel adhesion protein (MAP) anchor and LL-37, an antimicrobial peptide. MAPs can selectively attach to metal and organic surfaces via L-3,5-dihydroxyphenylalanine (L-DOPA), a nonstandard amino acid that was incorporated using a genetically recoded organism (GRO). Because this peptide is toxic to the GRO in which it is produced, we designed a better controlled inducible system that limits basal expression. This was achieved through a novel T7 riboregulation system that controls expression at both the transcriptional and translational levels.
LIC
Biofilm formation on surfaces is an issue in the medical field, naval industry, and other areas. We developed an anti-fouling peptide with two modular components: a mussel adhesion protein (MAP) anchor and LL-37, an antimicrobial peptide. MAPs can selectively attach to metal and organic surfaces via L-3,5-dihydroxyphenylalanine (L-DOPA), a nonstandard amino acid that was incorporated using a genetically recoded organism (GRO). Because this peptide is toxic to the GRO in which it is produced, we designed a better controlled inducible system that limits basal expression. This was achieved through a novel T7 riboregulation system that controls expression at both the transcriptional and translational levels.
ELIC
Biofilm formation on surfaces is an issue in the medical field, naval industry, and other areas. We developed an anti-fouling peptide with two modular components: a mussel adhesion protein (MAP) anchor and LL-37, an antimicrobial peptide. MAPs can selectively attach to metal and organic surfaces via L-3,5-dihydroxyphenylalanine (L-DOPA), a nonstandard amino acid that was incorporated using a genetically recoded organism (GRO). Because this peptide is toxic to the GRO in which it is produced, we designed a better controlled inducible system that limits basal expression. This was achieved through a novel T7 riboregulation system that controls expression at both the transcriptional and translational levels.
Testing DNA Constructs
Time flies. Luckily, you're the pilot.
Testing Promoters
Biofilm formation on surfaces is an issue in the medical field, naval industry, and other areas. We developed an anti-fouling peptide with two modular components: a mussel adhesion protein (MAP) anchor and LL-37, an antimicrobial peptide. MAPs can selectively attach to metal and organic surfaces via L-3,5-dihydroxyphenylalanine (L-DOPA), a nonstandard amino acid that was incorporated using a genetically recoded organism (GRO). Because this peptide is toxic to the GRO in which it is produced, we designed a better controlled inducible system that limits basal expression. This was achieved through a novel T7 riboregulation system that controls expression at both the transcriptional and translational levels.
Testing Beta-Homologs
Biofilm formation on surfaces is an issue in the medical field, naval industry, and other areas. We developed an anti-fouling peptide with two modular components: a mussel adhesion protein (MAP) anchor and LL-37, an antimicrobial peptide. MAPs can selectively attach to metal and organic surfaces via L-3,5-dihydroxyphenylalanine (L-DOPA), a nonstandard amino acid that was incorporated using a genetically recoded organism (GRO). Because this peptide is toxic to the GRO in which it is produced, we designed a better controlled inducible system that limits basal expression. This was achieved through a novel T7 riboregulation system that controls expression at both the transcriptional and translational levels.
Assembling Reporter/Recombinase Constructs
If opportunity doesn't knock, build a door.
Gibson Assembly
Biofilm formation on surfaces is an issue in the medical field, naval industry, and other areas. We developed an anti-fouling peptide with two modular components: a mussel adhesion protein (MAP) anchor and LL-37, an antimicrobial peptide. MAPs can selectively attach to metal and organic surfaces via L-3,5-dihydroxyphenylalanine (L-DOPA), a nonstandard amino acid that was incorporated using a genetically recoded organism (GRO). Because this peptide is toxic to the GRO in which it is produced, we designed a better controlled inducible system that limits basal expression. This was achieved through a novel T7 riboregulation system that controls expression at both the transcriptional and translational levels.
LIC
Biofilm formation on surfaces is an issue in the medical field, naval industry, and other areas. We developed an anti-fouling peptide with two modular components: a mussel adhesion protein (MAP) anchor and LL-37, an antimicrobial peptide. MAPs can selectively attach to metal and organic surfaces via L-3,5-dihydroxyphenylalanine (L-DOPA), a nonstandard amino acid that was incorporated using a genetically recoded organism (GRO). Because this peptide is toxic to the GRO in which it is produced, we designed a better controlled inducible system that limits basal expression. This was achieved through a novel T7 riboregulation system that controls expression at both the transcriptional and translational levels.
ELIC
Biofilm formation on surfaces is an issue in the medical field, naval industry, and other areas. We developed an anti-fouling peptide with two modular components: a mussel adhesion protein (MAP) anchor and LL-37, an antimicrobial peptide. MAPs can selectively attach to metal and organic surfaces via L-3,5-dihydroxyphenylalanine (L-DOPA), a nonstandard amino acid that was incorporated using a genetically recoded organism (GRO). Because this peptide is toxic to the GRO in which it is produced, we designed a better controlled inducible system that limits basal expression. This was achieved through a novel T7 riboregulation system that controls expression at both the transcriptional and translational levels.
Testing Promoter/Recombinase Constructs
Time flies. Luckily, you're the pilot.
Testing Promoters
Biofilm formation on surfaces is an issue in the medical field, naval industry, and other areas. We developed an anti-fouling peptide with two modular components: a mussel adhesion protein (MAP) anchor and LL-37, an antimicrobial peptide. MAPs can selectively attach to metal and organic surfaces via L-3,5-dihydroxyphenylalanine (L-DOPA), a nonstandard amino acid that was incorporated using a genetically recoded organism (GRO). Because this peptide is toxic to the GRO in which it is produced, we designed a better controlled inducible system that limits basal expression. This was achieved through a novel T7 riboregulation system that controls expression at both the transcriptional and translational levels.
Testing Beta-Homologs
Biofilm formation on surfaces is an issue in the medical field, naval industry, and other areas. We developed an anti-fouling peptide with two modular components: a mussel adhesion protein (MAP) anchor and LL-37, an antimicrobial peptide. MAPs can selectively attach to metal and organic surfaces via L-3,5-dihydroxyphenylalanine (L-DOPA), a nonstandard amino acid that was incorporated using a genetically recoded organism (GRO). Because this peptide is toxic to the GRO in which it is produced, we designed a better controlled inducible system that limits basal expression. This was achieved through a novel T7 riboregulation system that controls expression at both the transcriptional and translational levels.
Safety - Protocol from 2014
Researchers
Our laboratory is certified for Biosafety Level 1 (BS-1) work, and we have access to a Biosafety Level 2 lab for BSL2 work. Our work fell within the BSL-1 domain, as indicated by the Center of Disease Control guidelines. All biological waste was stored in autoclave bags and was autoclaved prior to disposal. Sharps and broken glassware were disposed of according to institutional guidelines. Hazardous liquid waste was clearly labeled, and stored in secondary containment for disposal by the institution. Thus, although there is potential for harm to researchers, it is minimized through following procedures approved and used by many laboratories at Yale. It is also minimized by training and common sense. E. coli strains used were common laboratory strains and not pathogenic.
- http://ehs.yale.edu/training/biosafetypartI
- http://ehs.yale.edu/training/biosafetypartII
- http://learn.caim.yale.edu/chemsafe/login.html
- http://ehs.yale.edu/training/hazardous-chemical-waste-management
All materials were used in accordance with local, national, and Yale Biosafety requirements. Standard lab practices were followed, including secondary containment of chemicals, proper storage of volatiles and flammables, and separation of acids and bases. Nitrile gloves were worn at all times within the lab. A pipet was kept exclusively for ethidium bromide use. Fume hoods were used when handling volatile compounds, concentrated acids and bases, and other reagents. Inhalation and skin contact was avoided. Chemical agents were properly disposed of in designated biohazard waste bins. When UV light was used to visualize gels or GFP, special care was taken to avoid skin or eye exposure. Absolutely no food was allowed in the lab.
Our project was overseen by the Yale Biological Safety Committee and the Office of Environmental Health and Safety (OEHS). Our project has been approved as consistent with Yale's safety regulations. No changes to our project were required since proper protocols were followed. Training was completed as described above.p
Public Safety
We do not anticipate any threat to public safety. Organisms worked with are all non-pathogenic. They are likely unable to survive outside of the lab environment, because they will be unable to compete with other organisms in nature. Biomaterials were autoclaved after use. We did not use gloves to touch doors outside of the laboratory to avoid others coming into contact with our chemical and biological agents.
Hands were washed before and after leaving the laboratory.
Our project will ideally be scaled up for medical and industrial use. Possible issues are allergies to the product and if used in immense qualities, DOPA toxicity to the environment. However, proteins and amino acids will be degraded and recycled by the environment, so we do not believe the product will be extremely harmful.
If we continued future work on this project we would assess the risks and hazards associated with it. Once understood, we would attempt to alter the design to minimize these risks. The E. Coli chassis for the product we are designing is not likely to be a great risk on its own.
Environmental Safety
There are no additional risks posed by our projects compared to other general BSL1 lab concerns. Our bacteria are not pathogenic and are unable to survive outside of the lab environment, because they are unable to effectively compete with other organisms in nature. They do not cause adverse reactions in immunocompetent humans. They do not cause infection. Toxic materials were all disposed of according to Yale waste standards to prevent adverse environmental impact.
There are no safety issues raised by the BioBrick parts submitted to the Registry this year. None of our constructs create toxic gene products for humans or animals.
Yale iGem 2015
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© Yale iGEM 2015