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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 (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)[2], 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[3] is an open-source primer-design software, and we modified its C codes in parts to design the BioBrick-Barcodes.
Experiment
Results
电泳照片。
