Team:Yale/results
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Developing a Framework for the Genetic Manipulation of Non-Model and Environmentally Significant Microbes
Project Results: Overview
Click on a component to learn more about our method.
Technique-Independent Procedures
MAGE Implementation Pathway
CRISPR-Cas9 Implementation Pathway
Growth
Cyanobacterium
The growth rate of PCC7002 was found to be highly sensitive to growth conditions, particularly light saturation and CO2 content of air. PCC7002 samples grown in CO2-supplemented conditions grew significantly faster (as measured by OD 730 nm) than samples grown in atmospheric conditions, and samples supplemented with 10mM glycerol grew to a much higher saturation than non-supplemented samples in the same period of time (glycerol supplement = top graph; no glycerol = middle graph). We also measured the ratio of chlorophyll content (OD 650 nm) to cell saturation (OD 730 nm) for cell populations grown in glycerol-supplemented versus nonsupplemented conditions. The glycerol was also found to not shunt cells away from photosynthetic pathways, as populations grown in glycerol had the same chlorophyll content per cell as nonsupplemented populations (bottom graph, PCC7002 grown in A+ medium is shown in green).
Transformation
Cyanobacterium
PCC7002 cell populations transformed with linear kanR genes flanked by 1000 bp homology arms up- and downstream of the gene demonstrated resistance to 200 µh/ml of kanamycin after a 24 hour incubation period and recovery in A+ medium. The six leftmost tubes in the picture below contain experimental samples which were recovered and inoculated in kanamycin-containing A+ medium; the four rightmost samples are all wild-type controls in kanamycin medium.
Selection
Cyanobacterium
PCC7002 was found to be susceptible to all antibiotics tested around standard E. coli concentrations, though light-sensitive antibiotics (tetracycline and rifampicin) were less effective due to degradation in light-saturated growth chambers. Click on the links below to see the growth curves for PCC7002 samples grown in different concentrations of tested antibiotics.
mutS Gene Knockout
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We successfully obtained mutS knockout genotypes of PCC7002 by transforming linear fragments for homologous recombination. Presumably due to the polyploidy of PCC7002 cells, however, we did not obtain homozygous ∆mutS mutants; doing so will require several more rounds of growth and plating for single colonies. Back to Top
DNA Assemblies
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
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.
Yale iGem 2015
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