From 2009.igem.org

Revision as of 12:45, 21 September 2009

Team:USTC/Tool

Introduction

Problem 1: How to distinguish the different biobricks in a biobrick-pool in experiment?

To take a DNA sequencing?---it's costly and time-consuming.

We are faced with such a problem to determine the final outputs of E.ADEM (E.coli Automatic Directed Evolution Machine)[1]. An inspiration comes from the supermarket checkout system where thousands of commodities are tagged by barcodes. This universal commercial ID both shortens the check-out time and adds the accuracy. By comparing our problem with this system, what we need to do can be concluded as to design barcodes for biobricks, BioBrick-Barcode, as we call it.

Problem 2: Where is the scanner?

PCR (Polymerase Chain Reaction)[http://en.wikipedia.org/wiki/PCR], which is so conveniently conducted, can serve as the scanner while the primers it needs are the analogue of barcode sequences.

Solution: To check the final outputs in the system of E.ADEM (E.coli Automatic Directed Evolution Machine), we need to design a set of DNA oligo-sequences primers as the barcodes to go through the scanner of PCR and help determining the final evolutionary products.

Design

A set of barcodes under a certain kind of barcode reader has to satisfy its conditions in order to be picked out respectively. For BioBrick-Barcodes, the oligo-sequences must meet several conditions as we considered:

1. Since the BioBrick-Barcodes function as PCR primers, they must satisfy the basic conditions as high-quality primers:

  a. Each primer should be 20-30 nucleotides in length; 
  b. Contain approximately equal numbers of 4 bases, with a balanced distribution of G&C residues;
  c. Hold a low propensity to form stable secondary structures;
  d. Forward and reverse primers can work properly together; 

2. As a set of barcodes, each of the primers should be perceivably different in order to be identified by the scanner:

  a. Any one of the primer cannot lead to the PCR of other DNA sequences(a plasmid with a certain target sequence.)
  b. All of the primers should be specific for the target sequence without combining with other sites. 

Software

Primer3[http://primer3.sourceforge.net/releases.php] is an open-source primer-design software, and we modified its C codes in parts to design the BioBrick-Barcodes. Since Primer3 is mainly used to pick primers from a DNA template while the BioBrick-Barcodes are supposed to be randomly produced, and a set of BioBrick-Barcodes should be diverse to avoid overlapping PCR, we changed several functions and structure of Primers.

  1. Add a function to randomly produce the DNA oligo-sequences which replaced the previous function used to 

Experiment

Results

电泳照片。

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