Team:British Columbia/Parts

Table of Contents

• Screening Vector
• Characterization of Screening Vector:
• Characterized Part

• PROJECT

  • 
  • Inspiration
  • Description
  • Approach
  • Hardware
  • Results
  • Parts
  • Future
• LABORATORY
  • 
  • Experimental Design
  • Protocols
  • Notebook
  • Safety
• MODEL
• HUMAN PRACTICES

  • 
  • Integrated Human Practices
  • Education/Outreach
  • Collaborations
• TEAM

  • 
  • Team
  • Attributions
  • Sponsors
• MEDALS

Screening Vector

pSB1C3 + B0030 + K895006 + J31008

Type: (according to igem)

Introduction: The vector-trap plasmid was designed to ensure the maximum possibility of acquiring a gene that may contain the promoter-transcription factor combination that we want. We were inspired by Uchiyama and Watanabe’s plasmid vector design, with multiple changes(1). By using a blunt end restriction site (NruI), we ensured that a higher variety of DNA would be ligated. We compensated for the lower ligation efficiency through fast-link DNA ligase and blunt-end fixing for the prepped metagenomic DNA. As per the paper, a GFP gene was added for possible FACs screening, but due to blunt ends ligate with different orientations, a reverse RFP was added as well. The reporter genes allowed FACs to happen for high-throughput screening of the fluorescing cells if a promoter were to be ligated into the vector.

Purpose:

The vector trap for screening use

Procedure:

See more in experiment page (SIGEX)

Result: See SIGEX result on the Results page

Reference: Uchiyama, T., & Watanabe, K. (2008). Substrate-induced gene expression (SIGEX) screening of metagenome libraries. Nature protocols, 3(7), 1202.

Characterization of Screening Vector

pSB1C3 + B0030 + K895006 + J31008 + K880005 (without rbs)

Purpose: To verify the functionality of the vector for metagenomic DNA screening

Result:

Figures 1 and 2. Normalized fluorescence values of controls and samples were determined over a period of 16 hours. Samples were transformed with a GFP-Promoter capable of recognizing transcription factors that can bind to saxitoxin. Various levels of saxitoxin (ppm) were administered to screen cells with potential transcription factors based on the relative fluorescence readout.

Conclusion: With the positive result from the final test assuming that there are promoters in the vector, it is likely that the screening vector is functioning as expected.

Characterized Part

Purpose: To assist the testing of our hardware device

Introduction: We digested the promoter responsive to Lead (BBa_I721001) with EcoRI and SpeI and digested the GFP plasmid (BBa_I13401) with XbaI and PstI. The promoter and reporter were Ampicillin resistant. These digested fragments were mixed and ligated to the provided, linearized pSB1C3 plasmid. The ligation mix was grown under Chloramphenicol selection. The resulting colonies were tested for responsive GFP production following the addition of Lead Nitrate.

Result see the registry