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Team:USTC/Tool
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Team:USTC/Tool
Contents |
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. 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), 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.
Experiment
