From 2009.igem.org
Team:USTC/Tool
Problem & Solution
- 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 identify 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)[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, we need to design a set of DNA oligo-sequences primers as the barcodes to go through the scanner of PCR and help identify 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.
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 three functions and structure of Primers.
1. Add a function to randomly produce the DNA oligo-sequences which replaced the previous function used to pick
segments from the template.
2. Modified the structure used to store information of each primer.
3. Use the Libray_Mispriming to choose diverse primers.
We design a set of ten diverse primers by using the modified software. Apparently, we need to conduct experiments to test its feasibility.
1.Synthesize the DNA oligo-sequence;
2.Construct plasmids;
3.Conduct a 10*10 cross experiment: Each primer reacts with all the plasmids;
Expectation: only complementary couple leads to PCR results.
Results
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| In the electrophoretogram, only the combination of primer 5 & plasmid 5, primer 6 & plasmid 6, primer 7 & plasmid 7 lead to PCR results. Along with other electrophoretograms we conclude that this set of primers can be used as a set of barcodes.
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Application
- In Synthetic Biology: BioBrick barcodes are a set of artifical DNA parts designed for PCR. They can be used alone as a high-quality PCR primer. Also, they can be used in a group as a convenient tool to identify the biobrick parts just as we do.
- Beyond Synthetic Biology: We create a new software to design PCR primers without a template. The primers chosen from a random oligo-sequences can enlarge the application scope of PCR.
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