Team:British Columbia/Experiments

Table of Contents

• SIGEX
  • Screening Plasmid
  • Metagenomic Library
  • Electroporation
  • Cell Sorting
  • Efficacy Test
• Screening The Pre-existing E.coli Promoter Library
• OD Growth Curve
• Screening with Saxitoxin

• 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

Substrate-Induced Gene Expression (SIGEX)

Purpose

Screening metagenomic DNA extracted from organisms co-existing with harmful algal blooms for transcription factors that respond to saxitoxin.

Justification

Little information about the transcription factors that respond to saxitoxin is found in the literature.

As saxitoxin (STX) effectively inhibits neurons, it is not harmful to prokaryotes and those organisms can survive in the presence of it, so it is possible that STX can regulate their genomic activities.

General Procedure

Screening Vector Construction

Our vector was designed to yield the highest possible chance of inducing a measurable response from an inserted section of metagenomic DNA. Fluorescence was used as a means of quantifying the strength of a STX response due to the fluorescence activated cell sorting equipment available to us. Our vector was also designed to be able to report a fluorescence signal regardless of the way the blunt-end fixed insert was inserted by having fluorescence genes (RFP and GFP) on either side of the insert. Our full vector design can be seen:

[Figure1: pSB1C3 vector added with forward rbs+GFP and reverse rbs+GFP, and a NruI cut side in between. More about the vector can be seen on our Parts page]

The vector was also designed with a NRU1 cut site for insertion of the metagenomic fragments. After the vector was digested, it was treated with rSAP to dephosphorylate the 5’ and 3’ end to minimize self ligation.

Metagenomic DNA Extraction

Based on algal bloom data from the CFIA, metagenomic DNA from environments known to have contained high concentrations of STX at the time samples were taken were then prepared for insertion into the screening vector. The DNA was first sheared to sizes of around 6kb and then blunt-end fixed. 6kb fragments of metagenomic DNA were subsequently ligated into the screening vector.

Transformation and Colony Collection:

Vector-insert pairs were transformed into E.coli by electroporation to yield the highest possible amount of colonies. Colonies were then counted and pooled via scraping where they would be frozen down until cell sorting

Cell Sorting

The library of E.coli cells containing vector-insert pairs were sorted using a fluorescence activated cell sorter. A first uninduced sort was always done to remove and constitutively expressed cells. Cells were then induced with 2ppm STX, sorted again and then the light fraction was pooled and plated. Colonies were inoculated into 96 well plates and induced again. Using a plate reader, fluorescence was measured and the clones that showed the highest relative fluorescence were noted.

Efficacy Test

The clones that showed the most STX induced fluorescence were plated in 384-well plates in replicates of 3 and in varying STX concentrations. The data showed an increasing trend of fluorescence with an increase is STX concentration within the range of 0.4ppm - 2ppm. In light of these positive results, we have sent in these samples for sequencing and our team is waiting on the results. We plan to do further efficacy test of the STX-inducible clones to further characterize the biosensor as well as data analysis on the DNA sequences.

Screening The Pre-existing E.coli Promoter Library

Purpose

Screening the pre-existing library for finding a promoter that can respond to STX

Justification

In previous studies, promoters that can interact with the substances that do not normally find in the E. coli habitat have been identified from the pre-existing E. coli promoter library

To increase our chance of finding a promoter that can interact with STX

General Procedure

Library Preparation

A pre-existing E.coli K12 promoter library[1] was replicated from 384 master plates and subsequently transferred to 1536 well plates using a liquid handler. 1536 well plates were used in order to decrease the amount of STX used in the experiments as it is expensive. We could also be confident the experiment could be carried out accurately due to the high accuracy of the liquid handler. An LB media contain 2ppm STX was added to each well in order to induce any potential STX-inducible promoters. A concentration of 2ppm was chosen as we did not want to miss any potential hits that would not be sensitive enough to be detectable at lower levels such as the CFIA’s testing standards.

[Figure2: The white plates on the left contain the media (LB, LB+STX, LB+HCl (control)) that were loaded into the wells on the 1536 microwell plates. The black plates on the right are black bottom 1536 well-plates.]

Screening:

Screening was conducted by a robotic workstation that stored the 1536-well plates in an incubator and removed them once every hour to measure fluorescence over the course of approximately 24 hours.