Team:Calgary/Project/Achievements

From 2014.igem.org

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<li>Sporulated <i>B. subtilis</i></li>
<li>Sporulated <i>B. subtilis</i></li>
<li>Activated <i>B. subtilis</i> spores back to vegetative state</li>
<li>Activated <i>B. subtilis</i> spores back to vegetative state</li>
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</ul>
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<li>Isolated the master regular for competency, <i>comK</i> from the genome of <i>B. subtilis</i>  
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<h2>Detectives:</h2>
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<ul>
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<li>Constructed a reporter operon consisting of a
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constitutive promoter (Pveg), ribosome binding site, operator (C1434) and reporter (LacZ/RFP).</li>
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<li>Constructed a complementary repressor operon
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consisting of a constitutive promoter (Pveg), ribosome
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binding site, and repressor pertaining to the operator.
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Repressor was also flanked with regions homologous to certain
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sequences within our pathogens of interest.</li>
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<li>Isolated the comK gene from the genome of <i>B. subtilis</i>  
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using PCR and inserted it into a Biobrick vector for future application  
using PCR and inserted it into a Biobrick vector for future application  
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as an iGEM standard part. The vast majority of my work was on the genetic
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as an iGEM standard part.  
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circuit, which was done under Dan. You should probably ask him about the
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specifics since he spearheaded it throughout the summer and concluded it in October.</li>
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<li>Biobricked and submitted the full-length <i>lacZ</i> gene, previously non-existant in the latest  
<li>Biobricked and submitted the full-length <i>lacZ</i> gene, previously non-existant in the latest  
distribution plates</li>
distribution plates</li>
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<li>Constructed a reporter operon consisting of a
 +
constitutive promoter (Pveg), ribosome binding site, operator (C1434) and reporter (LacZ/RFP).</li>
 +
<li>Constructed a complementary repressor operon
 +
consisting of a constitutive promoter (Pveg), ribosome
 +
binding site, and repressor pertaining to the operator.
 +
Repressor was also flanked with regions homologous to certain
 +
sequences within our pathogens of interest.</li>
</ul>
</ul>

Revision as of 18:45, 17 October 2014

Achievements

Policies and Practices:

  • Thoroughly researched our target markets and differing legislations that could affect our system
  • Connected with many different experts in the fields of infectious diseases, anti-malarial medications and public health to truly understand the needs and feasibility of the project
  • Connected with the foremost authority in low cost diagnostics, Foundation for Innovative New Diagnostics (FIND), to discuss the viability of our design and concept
  • Connected with a small village clinic in rural Uganda, which gave an opportunity to see the facility as well as discuss the topic with local physicians
  • Featured iGEM and our project on a global stage at the United Nations, in Geneva, and created discussion regarding the advancement of synthetic biology

Modelling:

  • Designed and manufactured a prototype of informed design based on our biological system
  • Cost analysis of our prototype and system to ensure that this project is feasible in developing nations
  • Quantified the visible threshold of detection for several reporters using time-lapsed cell count and absorbance readings, as well as a programmed colour sensor
  • Programmed an Arduino UNO microcontroller, colour sensor, and LCD screen to detect colour change among detection chambers, allowing for quantification of the colour
  • Applied circuitry of a heating pad/temperature considerations for ideal isothermal PCR conditions
  • Used Autodesk Maya Software to visually model and represent our system
  • Used Solid-works Software to model fluid flow analysis for different prototype designs

Transformation:

  • Transformed B. subtilis with a primer product containing 125 bp of homologous sequence flanking our gene of interest
  • Modified and optimized general transformation: characterized effect of changing xylose concentrations, temperature, and shaking?
  • Optimization of transformation in small, liquid volume
  • Sporulated B. subtilis
  • Activated B. subtilis spores back to vegetative state
  • Isolated the master regular for competency, comK from the genome of B. subtilis using PCR and inserted it into a Biobrick vector for future application as an iGEM standard part.

Sample Preparation:

  • Demonstrated efficacy of isothermal PCR to amplify DNA sequences without the need of a thermocycler, using freeze-dried components
  • Demonstrated ability to amplify target DNA sequence from whole E. coli cells in a blood mixture
  • Obtained specificity in target sequence primers - specific to numerous strains of N. meningititis but not other pathogens including a commensal strain of N. meningititis

Detection/Genetic circuit

  • Designed composite regulatory promoter sequences - used to allow repression of a downstream reporter with a specific repressor. Includes 5 variants of repressible promoters in either single or triplicate repeats for variation in strength of repression
  • Demonstrated interaction (i.e. repression) between the reporter gene and repressor gene in E. coli
  • Demonstrated the efficacy of homologous recombination in B. subtilis and showed the minimum length of flanking sequences
  • Biobricked and submitted the full-length lacZ gene, previously non-existant in the latest distribution plates
  • Constructed a reporter operon consisting of a constitutive promoter (Pveg), ribosome binding site, operator (C1434) and reporter (LacZ/RFP).
  • Constructed a complementary repressor operon consisting of a constitutive promoter (Pveg), ribosome binding site, and repressor pertaining to the operator. Repressor was also flanked with regions homologous to certain sequences within our pathogens of interest.