Difference between revisions of "Team:Yale/results"

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           <h2>Cyanobacterium</h2>
 
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           <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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           <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).
 
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           <p> 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.
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          <div class="launch__modal"><a href="#" data-reveal-id="transformOne" class="custom__button">Further Reading</a></div>
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           <p> 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.
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           <div class="launch__modal"><a href="#" data-reveal-id="selectOne" class="custom__button">Further Reading</a></div>
 
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       <h2 id="mage1">Gene Knockouts with Mage</h2>
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       <h2 id="mage1">mutS Gene Knockout</h2>
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      <h3>FLP-FRT Recombination</h3>
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       <p>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 <span class="return"> <a href="#top"> Back to Top</a></span></p>
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       <p> We successfully obtained <i>mutS</i> 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 <i>∆mutS</i> mutants; doing so will require several more rounds of growth and plating for single colonies. <span class="return"> <a href="#top"> Back to Top</a></span>
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       <h2 id="mage2">DNA Assemblies</h2>
 
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      <h3>If opportunity doesn't knock, build a door.</h3>
 
 
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Revision as of 03:03, 19 September 2015


<!DOCTYPE html> Yale iGem 2015: Results

Developing a Framework for the Genetic Manipulation of Non-Model and Environmentally Significant Microbes

Overview

Methods

Results

Modeling

Parts

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).

Sinorhizobium

Further Reading

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.

Sinorhizobium

Further Reading

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.

Further Reading

Sinorhizobium

Further Reading

mutS Gene Knockout

Lorem ipsum dolor sit amet, consectetur adipisicing elit. Neque magni ipsam veniam est obcaecati optio iure animi, minima nulla distinctio, rem dolor fugiat ea, aliquid dolorum aliquam cumque. Error, delectus!

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

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

Main Campus:

Yale Department of Molecular, Cellular & Developmental Biology

Attn: Farren Isaacs/iGEM

219 Prospect St

PO Box 208103

New Haven, CT 06520

Tel. +1(203) 432-3783

E-mail: igem.yale@gmail.com

© Yale iGEM 2015