Difference between revisions of "Team:Yale/results"
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| − | <p> | + | <p> 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). PCC7002 grown in A+ medium is shown in green. 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. |
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Revision as of 02:47, 19 September 2015
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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). PCC7002 grown in A+ medium is shown in green. 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.
Transformation
Cyanobacterium
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Selection
Cyanobacterium
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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.
Yale iGem 2015
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© Yale iGEM 2015